Pharmaceutical for oral delivery comprising MGBG

ABSTRACT

Disclosed herein are new oral pharmaceutical compositions of MGBG and related polyamine analogs, polyamine biosynthesis inhibitors, polyamine inhibitors of AMD-I and regulators of osteopontin, and their application for the treatment of disease.

This application claims the benefit of priority of U.S. provisionalapplications No. 61/226,060, filed Jul. 16, 2009, and No. 61/290,095,filed Dec. 24, 2009, the disclosures of which are incorporated byreference as if written herein in their entireties.

This invention was made with government support under Grant Number5U19MH081835-02 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

Disclosed herein are new oral pharmaceutical compositions of polyamineanalogs, polyamine biosynthesis inhibitors, polyamine inhibitors ofAMD-I and regulators of osteopontin, and their application for thetreatment of conditions including those modulated by osteopontin orassociated with elevated levels or activity of osteopontin, and thoserelated to viral infections such as human immunodeficiency virus (HIV).

MGBG (methylglyoxal bis(guanylhydrazone); mitoguazone) is a competitivepolyamine inhibitor of S-adenosyl methionine decarboxylase (AMD-I),which catalyzes the synthesis of spermidine, a polyamine. Theamino-acid-derived polyamines have long been associated with cell growthand cancer, and specific oncogenes and tumor-suppressor genes regulatepolyamine metabolism. Inhibition of polyamine synthesis has proven to begenerally ineffective as an anticancer strategy in clinical trials, butit is a potent cancer chemoprevention strategy in preclinical studies.Despite its novel mechanism of action and promising preclinical data,initial clinical trials of MGBG were ceased in the middle of 1960s dueto severe toxicity especially to self-renewing normal tissues such asthe bone marrow and intestinal tract (particularly severe mucositis),which were both dose and schedule dependent.

Regardless, research has continued with MGBG. A number of studies haveexamined potential uses in combination with other chemotherapeuticagents and innovative dosing regimens, designed to minimize side effectsand dose where possible. Others have focused on elucidating MGBG's modesof action in the body. Yet others have investigated MGBG's activity indiseases other than cancer.

Perhaps due to negative clinical findings in these early studies, todate, MGBG has been confined to intravenous use. As a practical matter,this presents a number of problems for treatment of many diseases,particularly of chronic or recurrent conditions. Administration via IVinjection or infusion must be done by a medical professional in ahospital setting. This not only presents an inconvenience and increasedcost to the subject, but it also exposes him or her to hospital-basedinfections and illnesses, this latter both from venipuncture and thehospital or clinic visit itself. In immunocompromised individuals suchas, for example, those with HIV or AIDS, individuals undergoingtreatment with immune system suppressors, and the elderly, this is arelevant concern. Thus, a subject with a long-term chronic conditionsuch as an autoimmune or hyperproliferative disorder, or a doctortreating such a subject, might find the cost, inconvenience, and risksof such a treatment more important than any potential therapeuticbenefits the drug might offer.

An oral formulation of MGBG, in contrast, would present severalbenefits. First, an oral formulation, for example a simple pill ortablet, may be taken outside of a hospital setting, increasing thepotential for subject ease of use and compliance. This permits a subjectto avoid infection risks concomitant with IV administration and hospitalvisits. Where early treatment can prevent the development of diseasecomplications, this is of particular benefit. Chronic low-doseadministration of MGBG is practically impossible in an IV formulation.Additionally, oral delivery typically avoids the high concentration peakand rapid clearance associated with an IV bolus dose. Yet anotheradvantage of an oral drug would be the ability to formulate MGBG as acombination composition with one or more other therapeutic agents.

Accordingly, disclosed herein are oral pharmaceutical formulations ofMGBG and other polyamine analogs, polyamine biosynthesis inhibitors,polyamine inhibitors of AMD-I and regulators of osteopontin, Alsodisclosed are methods for the treatment of diseases comprising theadministration of MGBG and other polyamine analogs, polyaminebiosynthesis inhibitors, polyamine inhibitors of AMD-I and regulators ofosteopontin.

Additionally, methods for the treatment of pain comprising theadministration of MGBG and other polyamine analogs, polyaminebiosynthesis inhibitors, polyamine inhibitors of AMD-I and regulators ofosteopontin are disclosed.

Accordingly, provided herein is a pharmaceutical composition for oraldelivery, comprising a polyamine analog or polyamine biosynthesisinhibitor together with at least one pharmaceutically acceptable oralexcipient.

Also provided herein is an oral pharmaceutical composition, comprisingpolyamine analog or polyamine biosynthesis inhibitor together with atleast one oral pharmaceutically acceptable excipient, which yields atherapeutically effective systemic plasma polyamine analog or polyaminebiosynthesis inhibitor level when orally administered to a subject.

Also provided herein is an oral pharmaceutical composition, comprisingpolyamine analog or polyamine biosynthesis inhibitor together with atleast one oral pharmaceutically acceptable excipient, which yields atherapeutically effective systemic plasma polyamine analog or polyaminebiosynthesis inhibitor level for the treatment of pain when orallyadministered to a subject.

In certain embodiments, the polyamine analog or polyamine biosynthesisinhibitor is a compound disclosed herein.

In certain embodiments, the polyamine analog or polyamine biosynthesisinhibitor is one known in the art.

In certain embodiments, the pharmaceutical composition yields atherapeutically effective systemic plasma level of a polyamine analog orpolyamine biosynthesis inhibitor for a period of at least 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 30, 36,or 48 hours. In further embodiments, the pharmaceutical compositionyields a therapeutically effective systemic plasma level of a polyamineanalog or polyamine biosynthesis inhibitor for at least a 6-hour period.In further embodiments, the pharmaceutical composition yields atherapeutically effective systemic plasma level of a polyamine analog orpolyamine biosynthesis inhibitor for at least a 12-hour period. Infurther embodiments, the pharmaceutical composition yields atherapeutically effective systemic plasma level of a polyamine analog orpolyamine biosynthesis inhibitor for at least an 18-hour period. Infurther embodiments, the pharmaceutical composition yields atherapeutically effective systemic plasma level of a polyamine analog orpolyamine biosynthesis inhibitor for at least a 24-hour period.

In certain embodiments, the pharmaceutical composition yields a plasmalevel of a polyamine analog or polyamine biosynthesis inhibitor of atleast 25, 50, 55, 60, 65, 75, 80, 85, 90, or 95 percent of the peakplasma concentration for at least 4 hours. In certain embodiments, thepharmaceutical composition yields a plasma level of a polyamine analogor polyamine biosynthesis inhibitor of at least 75% of the peak plasmaconcentration for at least 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24hours. In certain embodiments, the pharmaceutical composition yields aplasma level of a polyamine analog or polyamine biosynthesis inhibitorof at least 75% of the peak plasma concentration for at least 4 hours.In certain embodiments, the pharmaceutical composition yields a plasmalevel of a polyamine analog or polyamine biosynthesis inhibitor of atleast 75% of the peak plasma concentration for at least 6 hours. Incertain embodiments, the pharmaceutical composition yields a plasmalevel of a polyamine analog or polyamine biosynthesis inhibitor of atleast 75% of the peak plasma concentration for at least 8 hours. Incertain embodiments, the pharmaceutical composition yields a plasmalevel of a polyamine analog or polyamine biosynthesis inhibitor of atleast 50% of the peak plasma concentration for at least 8 hours. Incertain embodiments, the pharmaceutical composition yields a plasmalevel of a polyamine analog or polyamine biosynthesis inhibitor of atleast 50% of the peak plasma concentration for at least 12 hours. Incertain embodiments, the pharmaceutical composition yields a plasmalevel of a polyamine analog or polyamine biosynthesis inhibitor of atleast 50% of the peak plasma concentration for at least 18 hours. Incertain embodiments, the pharmaceutical composition yields a plasmalevel of a polyamine analog or polyamine biosynthesis inhibitor of atleast 25% of the peak plasma concentration for at least 18 hours. Infurther embodiments, the peak plasma concentration is a therapeuticallyeffective concentration. In yet further embodiments, the percentage ofpeak plasma concentration is therapeutically effective over the giventime period.

In certain embodiments, the pharmaceutical composition comprising thepolyamine analog or polyamine biosynthesis inhibitor has an oralbioavailability of at least 10, 20, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 45, 50, 55, or 60 percent. In furtherembodiments, the pharmaceutical composition has an oral bioavailabilityof at least 10%, 20%, 25%, 30%, 35%, 40%, or 45%. In furtherembodiments, the pharmaceutical composition has an oral bioavailabilityof at least 30%, at least 35%, at least 40% or at least 45%. In certainembodiments, the pharmaceutical composition has an oral bioavailabilityof at least 20%. In certain embodiments, the pharmaceutical compositionhas an oral bioavailability of at least 30%. In certain embodiments, thepharmaceutical composition has an oral bioavailability of at least 35%.In certain embodiments, the pharmaceutical composition has an oralbioavailability of at least 40%. In certain embodiments, thepharmaceutical composition has an oral bioavailability of at least 45%.In certain embodiments, the pharmaceutical composition has an oralbioavailability which yields a therapeutically effective plasma level ofa polyamine analog or polyamine biosynthesis inhibitor for at least a 24hour period in the subject with once-daily dosing. In certainembodiments, the pharmaceutical composition has an oral bioavailabilitywhich yields a therapeutically effective plasma level of a polyamineanalog or polyamine biosynthesis inhibitor for at least a 24 hour periodin the subject with twice-daily dosing. In certain embodiments, thepharmaceutical composition has an oral bioavailability which yields atherapeutically effective plasma level of a polyamine analog orpolyamine biosynthesis inhibitor for at least a 24 hour period in thesubject with thrice-daily dosing.

In certain embodiments, the pharmaceutical composition comprising thepolyamine analog or polyamine biosynthesis inhibitor has a half life ofat least 4, 6, 8, 10, 12, 14, 16, 18, 19, 20, 21, 22, 23, 24, 26, 28,30, or 36 hours. In certain embodiments, the pharmaceutical compositionhas a half life of at least 12 hours. In further embodiments, thepharmaceutical composition has a half life of at least 18 hours. Infurther embodiments, the pharmaceutical composition has a half life ofat least 20 hours. In further embodiments, the pharmaceuticalcomposition has a half life of at least 24 hours. In certainembodiments, the pharmaceutical composition has a half life of at least48, 72, 96, or 120 hours.

Additionally provided herein is a pharmaceutical composition for oraldelivery, comprising MGBG together with at least one pharmaceuticallyacceptable oral excipient.

Also provided herein is an oral pharmaceutical composition, comprisingMGBG together with at least one oral pharmaceutically acceptableexcipient, which yields a therapeutically effective systemic plasma MGBGlevel when orally administered to a subject.

Also provided herein is an oral pharmaceutical composition, comprisingMGBG together with at least one oral pharmaceutically acceptableexcipient, which yields a therapeutically effective systemic plasma MGBGlevel for the treatment of pain when orally administered to a subject.

In certain embodiments, the pharmaceutical composition yields atherapeutically effective systemic plasma MGBG level for a period of atleast 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 30, 36, or 48 hours. In further embodiments, thepharmaceutical composition yields a therapeutically effective systemicplasma MGBG level for at least a 6-hour period. In further embodiments,the pharmaceutical composition yields a therapeutically effectivesystemic plasma MGBG level for at least a 12-hour period. In furtherembodiments, the pharmaceutical composition yields a therapeuticallyeffective systemic plasma MGBG level for at least an 18-hour period. Infurther embodiments, the pharmaceutical composition yields atherapeutically effective systemic plasma MGBG level for at least a24-hour period.

In certain embodiments, the pharmaceutical composition yields a plasmalevel of MGBG of at least 25, 50, 55, 60, 65, 75, 80, 85, 90, or 95percent of the peak plasma concentration for at least 4 hours. Incertain embodiments, the pharmaceutical composition yields a plasmalevel of MGBG of at least 75% of the peak plasma concentration for atleast 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 hours. In certainembodiments, the pharmaceutical composition yields a plasma level ofMGBG of at least 75% of the peak plasma concentration for at least 4hours. In certain embodiments, the pharmaceutical composition yields aplasma level of MGBG of at least 75% of the peak plasma concentrationfor at least 6 hours. In certain embodiments, the pharmaceuticalcomposition yields a plasma level of MGBG of at least 75% of the peakplasma concentration for at least 8 hours. In certain embodiments, thepharmaceutical composition yields a plasma level of MGBG of at least 50%of the peak plasma concentration for at least 8 hours. In certainembodiments, the pharmaceutical composition yields a plasma level ofMGBG of at least 50% of the peak plasma concentration for at least 12hours. In certain embodiments, the pharmaceutical composition yields aplasma level of MGBG of at least 50% of the peak plasma concentrationfor at least 18 hours. In certain embodiments, the pharmaceuticalcomposition yields a plasma level of MGBG of at least 25% of the peakplasma concentration for at least 18 hours. In further embodiments, thepeak plasma concentration is a therapeutically effective concentration.In yet further embodiments, the percentage of peak plasma concentrationis therapeutically effective over the given time period.

In certain embodiments, the pharmaceutical composition comprising MGBGhas an oral bioavailability of at least 10, 20, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 55, or 60 percent. Infurther embodiments, the pharmaceutical composition has an oralbioavailability of at least 10%, 20%, 25%, 30%, 35%, 40%, or 45%. Infurther embodiments, the pharmaceutical composition has an oralbioavailability of at least 30%, at least 35%, at least 40% or at least45%. In certain embodiments, the pharmaceutical composition has an oralbioavailability of at least 20%. In certain embodiments, thepharmaceutical composition has an oral bioavailability of at least 30%.In certain embodiments, the pharmaceutical composition has an oralbioavailability of at least 35%. In certain embodiments, thepharmaceutical composition has an oral bioavailability of at least 40%.In certain embodiments, the pharmaceutical composition has an oralbioavailability of at least 45%. In certain embodiments, thepharmaceutical composition has an oral bioavailability which yields atherapeutically effective plasma level of MGBG for at least a 24 hourperiod in the subject with once-daily dosing. In certain embodiments,the pharmaceutical composition has an oral bioavailability which yieldsa therapeutically effective plasma level of MGBG for at least a 24 hourperiod in the subject with twice-daily dosing. In certain embodiments,the pharmaceutical composition has an oral bioavailability which yieldsa therapeutically effective plasma level of MGBG for at least a 24 hourperiod in the subject with thrice-daily dosing.

In certain embodiments, the pharmaceutical composition comprising MGBGhas a half life of at least 4, 6, 8, 10, 12, 14, 16, 18, 19, 20, 21, 22,23, 24, 26, 28, 30, or 36 hours. In certain embodiments, thepharmaceutical composition has a half life of at least 12 hours. Infurther embodiments, the pharmaceutical composition has a half life ofat least 18 hours. In further embodiments, the pharmaceuticalcomposition has a half life of at least 20 hours. In furtherembodiments, the pharmaceutical composition has a half life of at least24 hours. In certain embodiments, the pharmaceutical composition has ahalf life of at least 48, 72, 96, or 120 hours.

Also provided herein is a pharmaceutical composition comprising MGBGtogether with at least one oral pharmaceutically acceptable excipient,which yields a therapeutically effective systemic plasma MGBG level whenorally administered to a subject, which does not have substantiallydose-limiting side effects. In certain embodiments, said side effectsare gastrointestinal. In further embodiments, said gastrointestinal sideeffects are chosen from nausea, vomiting, diarrhea, abdominal pain, oralmucositis, oral ulceration, pharyngitis, stomatitis, andgastrointestinal ulceration. In further embodiments, saidgastrointestinal side effects are chosen from inhibition ofgastrointestinal mucosal proliferation, inhibition of migration ofdeveloping epithelial lumen cells, and inhibition of differentiation ofstem or progenitor cells into epithelial lumen cells. In certainembodiments, said side effects are chosen from thrombocytopenia,leukopenia, phlebitis, laryngitis, cellulitis, dermatitis, andhypoglycemia.

Also provided herein is a low-dose oral pharmaceutical composition forchronic delivery, comprising a therapeutically effective amount of MGBGand at least one pharmaceutically acceptable excipient, which does nothave substantial gastrointestinal side effects. In certain embodiments,the low-dose oral pharmaceutical composition for chronic delivery,comprising a therapeutically effective amount of MGBG and at least onepharmaceutically acceptable excipient, which does not have substantialgastrointestinal side effects, yields a therapeutically effective plasmalevel of MGBG for at least a 24 hour period in the subject withonce-daily dosing.

In certain embodiments, the pharmaceutical composition is formulated asa tablet or capsule. For example, in certain embodiments, thepharmaceutical composition comprises:

-   0.1-50% of a polyamine analog or a polyamine biosynthesis inhibitor;-   0.1-99.9% of a filler;-   0-10% of a disintegrant;-   0-5% of a lubricant; and,-   0-5% of a glidant.

In certain embodiments, the pharmaceutical composition comprises:

-   0.1-50% of MGBG;-   0.1-99.9% of a filler;-   0-10% of a disintegrant;-   0-5% of a lubricant; and,-   0-5% of a glidant.

In further embodiments,

-   said filler is chosen from a sugar, a starch, a cellulose, and a    poloxamer;-   said disintegrant is chosen from povidone and crospovidone;-   said lubricant is magnesium stearate; and-   said glidant is silicon dioxide.

In further embodiments,

-   said filler is chosen from lactose and microcrystalline cellulose;-   said disintegrant is chosen from povidone and crospovidone;-   said lubricant is magnesium stearate; and-   said glidant is silicon dioxide.

In certain embodiments, the pharmaceutical composition comprises:

-   10-300 mg of a polyamine analog or a polyamine biosynthesis    inhibitor, making up-   2-50% of the tablet content or capsule fill content;-   0-10% of a disintegrant;-   0-5% of a lubricant;-   0-5% of a glidant; and-   30-98% of a filler.

In certain embodiments, the pharmaceutical composition comprises:

-   10-300 mg of MGBG, making up 2-50% of the tablet content or capsule    fill content;-   0-10% of a disintegrant;-   0-5% of a lubricant;-   0-5% of a glidant; and-   30-98% of a filler.

In further embodiments, the pharmaceutical composition comprises

-   0.1-10% of a binder;-   0-5% of a surfactant;-   0-10% of an intergranular disintegrant; and-   0-10% of an extragranular disintegrant.

In further embodiments, the pharmaceutical composition may additionallycomprise

-   0-10% of a binder;-   0-5% of a surfactant;-   0-10% of an intergranular disintegrant; and-   0-10% of an extragranular disintegrant.

In further embodiments,

-   said binder is chosen from copolyvidone, hydroxypropyl-cellulose,    hydroxypropylmethylcellulose, and povidone;-   said surfactant is chosen from polyoxyethylene (20) sorbitan    monooleate, a poloxamer, and sodium lauryl sulfate;-   said intergranular disintegrant is chosen from croscarmellose    sodium, sodium starch glyconate, and crospovidone; and-   said extragranular disintegrant is chosen from croscarmellose    sodium, sodium starch glyconate, and crospovidone.

Also provided herein is a method of treating or delaying the onset ordevelopment of a condition in a subject in need thereof comprising theadministration of an oral pharmaceutical composition comprising MGBG andat least one pharmaceutically acceptable excipient. In certainembodiments, the oral pharmaceutical composition is delivered in atherapeutically effective amount. In certain embodiments, said oralpharmaceutical composition has an oral bioavailability of at least 30%.In certain embodiments, said oral pharmaceutical composition does nothave substantially dose-limiting side effects. In certain embodiments,the plasma level of MGBG is at least 75% of the peak plasmaconcentration for 4 or more hours. In further embodiments, said oralpharmaceutical composition yields a therapeutically effective systemicplasma MGBG level for at least a 12-hour period when orally administeredto a subject.

In certain embodiments, said condition is chosen from a proliferativedisorder, an inflammatory disease, and an autoimmune disease, andneuropathy. In certain embodiments, said condition is chosen fromrheumatoid arthritis, osteoarthritis, multiple sclerosis, HIVneuropathy, and HIV associated dementia.

In certain embodiments, said proliferative disorder is chosen from acancer, psoriasis, psoriatic arthritis and atopic dermatitis. In certainembodiments, the neuropathy is chosen from peripheral neuropathy,diabetic neuropathy, entrapment neuropathy (carpel tunnel syndrome),postherpetic neuralgia (PHN), chemotherapy-induced neuropathy, and HIVneuropathy.

In certain embodiments, the condition is chosen from a proliferativedisorder, rheumatoid arthritis, osteoarthritis, multiple sclerosis, HIVneuropathy, and HIV associated dementia. In certain embodiments, theproliferative disorder could, for example, be chosen from a cancer,psoriasis, psoriatic arthritis, and atopic dermatitis. In certainembodiments, the treatment results in decreased levels or activity ofosteopontin. In certain embodiments, the method further comprisesassaying for osteopontin level, and based on the level of osteopontin,administering an additional therapeutically effective amount of an oralpharmaceutical composition comprising MGBG and at least onepharmaceutically acceptable excipient.

Also provided is an oral pharmaceutical composition, comprising apolyamine analog or polyamine biosynthesis inhibitor together with atleast one oral pharmaceutically acceptable excipient, which yields atherapeutically effective systemic plasma level of the polyamine analogor polyamine biosynthesis inhibitor for the treatment of pain whenorally administered to a subject. Also provided is an oralpharmaceutical composition, comprising MGBG together with at least oneoral pharmaceutically acceptable excipient, which yields atherapeutically effective systemic plasma level of MGBG for thetreatment of pain when orally administered to a subject.

Also provided herein is a method of treatment of pain in a subject inneed thereof comprising the administration of a polyamine analog or apolyamine biosynthesis inhibitor, or a salt or protected derivativethereof. Also provided herein is a method of treatment of pain in asubject in need thereof comprising the administration of MGBG. Incertain embodiments, the MGBG is administered in a therapeuticallyeffective amount. Further provided is a method of treatment of pain in asubject in need thereof comprising the administration of atherapeutically effective amount of a pharmaceutical compositioncomprising MGBG and at least one pharmaceutically acceptable excipient.

In certain embodiments, the pain is chosen from inflammatory pain, paindue to nerve injury, chronic pain, intractable cancer pain, complexregional pain syndrome, neuropathic pain, surgical or post-surgicalpain, dental pain, pain resulting from dermal injury, lower back pain,headaches, migraine, tactile allodynia, and hyperalgesia. In certainembodiments, the pain is chronic. In other embodiments, the pain isacute. In certain embodiments, the pain is inflammatory pain.

In certain embodiments, the administration of MGBG or its pharmaceuticalcomposition is oral. In other embodiments, the administration isintravenous.

In certain embodiments, the administration is a combination of oral andintravenous. In certain embodiments, the first administration is oraland the second IV; in others the first is IV and the second oral; ineither case, additional oral or IV dosing may follow. In certainembodiments, the pain is surgical or post-surgical pain. For example, incertain embodiments, the pre-surgical administration is oral and theperi-surgical administration is IV; in others the pre-surgicaladministration is IV, the pre-surgical administration is also IV, andthe post-surgical administration is oral. In either case, additionaloral or IV dosing may follow. In certain embodiments, the pre-, peri-,and post-surgical administration is IV.

Also provided herein is a method of treatment of HIV neuropathy in asubject in need thereof comprising the administration of atherapeutically effective amount of an oral pharmaceutical compositioncomprising MGBG and at least one pharmaceutically acceptable excipient.In certain embodiments, the plasma level of MGBG is at least 75% of thepeak plasma concentration for 4 or more hours.

Also provided herein is a method of treatment of a condition in asubject in need thereof comprising the administration of

-   an oral pharmaceutical composition comprising MGBG and at least one    pharmaceutically acceptable excipient; and-   another therapeutic agent.

In certain embodiments, the MGBG is delivered in a therapeuticallyeffective amount. In other embodiments, the MGBG is delivered in asubtherapeutic amount. In certain embodiments, the other therapeuticagent is delivered in a therapeutically effective amount. In otherembodiments, the other therapeutic agent is delivered in asubtherapeutic amount. In certain embodiments, the MGBG and the othertherapeutic agent are delivered together in amounts which wouldindividually be subtherapeutic but which together are therapeuticallyeffective. In other embodiments, the MGBG and the other therapeuticagent are delivered together in amounts which are individuallytherapeutically effective.

Additionally provided herein are methods: of regulation of osteopontinactivity or levels in a subject as well as for treating or preventingconditions associated with an increased level or activity of osteopontinin a subject; of decreasing levels or the activity of osteopontin in acell, comprising contacting a cell with an effective amount of an agentthat inhibits S-adenosyl methionine decarboxylase (“AMD-I”), or inhibitspolyamine biosynthesis; of decreasing the level or activity ofosteopontin in a cell, comprising contacting the cell with an effectiveamount of MGBG, a salt of MGBG, or a protected derivative of MGBG; oftreating or preventing a condition associated with an increased level oractivity of osteopontin, comprising administering to a subject in needof such treatment an effective amount of an agent that inhibitsS-adenosyl methionine decarboxylase, or inhibits polyamine biosynthesisin the subject.

Additionally provided herein is a method of treating a condition. Themethod comprises administering to a subject in need of such treatment aneffective amount of MGBG, a salt of MGBG, a protected derivative ofMGBG, or a polyamine analog or polyamine biosynthesis inhibitor or asalt, a protected derivative, or a stereoisomer thereof, wherein thecondition is chosen from Crohn's disease, Parkinson's disease,inflammatory bowel disorder, multiple sclerosis (MS), amyotrophiclateral sclerosis (ALS), hepatitis, HBY, HCV, nephritis, cerebritis,glomerulonephritis, rheumatoid arthritis, type 2 diabetes, cardiacfibrosis and angiotensin type II associated hypertension, osteoporosis,a mast cell produced IgE mediated hypersensitivity immune reaction,peripheral sensory neuropathy associated with HIV infection or diabetesmellitus, asthma, autism, dermatomyositis, frailty, obesity, primarybiliary cirrhosis, primary sclerosing cholangitis, post-radiationsyndrome, psoriatic arthritis, sarcoidosis, scleroderma with or withoutpulmonary fibrosis, a kidney related autoimmune condition, diabeticnephropathy, a diabetic vascular complication, and a lymphoproliferationrelated autoimmune condition.

Additionally provided herein is a method of decreasing osteopontinsecretion from monocytes or macrophages, comprising contacting amonocyte or macrophage with an effective amount of an agent thatinhibits S-adenosyl methionine decarboxylase or inhibits polyaminebiosynthesis in the monocyte or macrophage. Further provided herein is amethod of decreasing osteopontin secretion from monocytes ormacrophages, comprising contacting a monocyte or macrophage with aneffective amount of MGBG or a salt or protected derivative thereof.

Additionally provided herein is a method of decreasing differentiationof macrophages from monocytes, comprising contacting a monocyte with aneffective amount of an agent that inhibits S-adenosyl methioninedecarboxylase or inhibits polyamine biosynthesis in the monocyte. Incertain embodiments the agent is MGBG, or a salt or protected derivativethereof.

The present invention provides a method of treating or preventing acondition associated with an increased level or activity of osteopontin.The method comprises administering to a subject in need of suchtreatment an effective amount of an agent that regulates the activity ofosteopontin. The condition can be any condition now known, or laterdiscovered, to be associated with an increased level or activity ofosteopontin. Examples of conditions associated with an increasedactivity of osteopontin include, but are not limited to, autoimmuneconditions, inflammatory conditions, neoplastic growth and tumormetastases. In one embodiment, the condition associated with anincreased level or activity of osteopontin is infiltration of immunecells to an affected area or increased level of CD14/CD16 macrophages ina subject.

In another embodiment, conditions associated with an increased activityof osteopontin include, but are not limited to, multiple sclerosis (MS),atherosclerosis and related coronary conditions, rheumatoid arthritis,lupus, nephritis, cerebritis, Crohn's disease, osteoporosis,inflammatory bowel disorder, breast cancer, ovarian cancer, pancreaticcancer, bladder cancer, lung cancer, colon cancer, gastric carcinomas,esophageal carcinomas, squamous cell carcinomas of the head or neck,prostate cancer, thyroid cancer, melanoma, kidney cancers, renal cellcarcinomas, endometrial cancer, small intestine cancer, duodenal cancer,cholangiocarcinoma, astrocytoma, AIDS lymphoma, follicular lymphoma,T-cell lymphoma, B-cell lymphoma, proliferative retinopathy,vitreoretinopathy, diabetic retinopathy, macular degeneration, non-HIVassociated dementia, HIV- and AIDS-associated dementia, focal segmentalglomerulosclerosis, membrane proliferative glomerulonephropathy,psoriasis, herpes virus associated disease, Castleman's disease,Kaposi's sarcoma, Alzheimer's disease, type 2 diabetes, cardiac fibrosisand angiotensin type II associated hypertension, mast cell produced IgEmediated hypersensitivity immune reactions, prelymphomatic orlymphoproliferation related autoimmune conditions, angioimmunoblasticlymphadenophathy (AILD), glomerulonephritis and other glomerulardiseases, immunoglobulin A (IgA) nephropathy, Amyotrophic LateralSclerosis (ALS), hepatitis including HBV and HCV, peripheral sensoryneuropathy associated with HIV infection or diabetes mellitus, asthma,autism, dermatomyositis, frailty, obesity, Parkinson's disease, primarybiliary cirrhosis, primary sclerosing cholangitis, post-radiationsyndrome, psoriatic arthritis, sarcoidosis, scleroderma with or withoutpulmonary fibrosis, kidney related autoimmune conditions, diabeticnephropathy and other diabetic vascular complications.

According to yet another aspect, the present invention provides a methodof decreasing osteopontin secretion from monocytes or macrophages. Themethod comprises contacting a monocyte or macrophage with an effectiveamount of an agent that regulates the activity of osteopontin.

According to yet another aspect, the present invention provides a methodof decreasing differentiation of macrophages from monocytes. The methodcomprises contacting a monocyte with an effective amount of an agentthat regulates the activity of osteopontin. In certain embodiments theagent that regulates the activity of osteopontin is MGBG, or a salt orprotected derivative thereof.

Also provided herein is a method of reducing viral load of an infectedsubject comprising administering to a subject infected by animmunodeficiency virus a therapeutically effective amount of a polyamineanalog or a polyamine biosynthesis inhibitor whereby reducing the viralload in the subject infected by the immunodeficiency virus. In certainembodiments the polyamine biosynthesis inhibitor is MGBG, or a salt orprotected derivative thereof.

Also provided herein is a method for treating an immunodeficiency viralinfection comprising administering to a subject infected by animmunodeficiency virus a therapeutically effective amount of a polyamineanalog or polyamine biosynthesis inhibitor and at least one an antiviralagent. Also provided herein is a method for preventing the onset of AIDSor conditions secondary to HIV infection comprising administering to asubject infected with human immunodeficiency virus a therapeuticallyeffective amount of a polyamine analog. In certain embodiments thepolyamine biosynthesis inhibitor is MGBG, or a salt or protectedderivative thereof.

Also provided herein is a pharmaceutical formulation which comprises apolyamine analog or polyamine biosynthesis inhibitor, an antiviralagent, and a pharmaceutically acceptable carrier, which may optionallybe provided as a kit. In certain embodiments, this pharmaceuticalformulation is in an amount of a single dose suitable to be taken by asubject in need of such treatment. In certain embodiments, saidtreatment is for a viral infection.

The agent useful in the methods of the invention can be any agent thatdecreases the activity of osteopontin. In one embodiment, the agent iscapable of inhibiting S-adenosyl methionine decarboxylase (“AMD-I”) orany pathway containing AMD I, e.g., any entity upstream or downstream ofa pathway containing AMD-I, especially any pathway containing AMD-I andassociated with adenosine production. In another embodiment the agent iscapable of inhibiting polyamine biosynthesis or any pathway involved inpolyamine biosynthesis. In general, a pathway containing AMD-I oradenosine is understood to refer to a pathway in which either AMD-I oradenosine is involved, including, for example, as a substrate, catalyst,product or by-product.

The agent can be any kind of known or later discovered agent that caninhibit the activity of the enzyme S-adenosyl methionine decarboxylase,can inhibit polyamine biosynthesis in, for example, a cell. In oneembodiment, the agent is a chemical agent, including, but not limitedto, organic molecules and salts, protected derivatives and stereoisomersthereof, inorganic molecules or various ionic or elemental entities.

Compounds for use in the methods and compositions disclosed hereininclude polyamine analogs and polyamine biosynthesis inhibitors, as wellas salts, prodrugs, solvates, anhydrous forms, protected derivatives,structural isomers, stereoisomers, amino acid conjugates, and porphyrinconjugates thereof. Any polyamine analog is suitable for use in themethods of the present invention.

Exemplary polyamine analogs used in the methods of the invention includecompounds of the structural formulas 1, 2, 3, 4, 5, 6, and 7 and thecorresponding stereoisomers, salts, and protected derivatives thereof.

Formula 1 has the structure

wherein

R₁, R₂, R₄, R₆ and R₇ are independently chosen from hydrogen, alkyl andaryl; and

R₃ and R₅, are alkyl groups.

Formula 2 has the structure

wherein

R₁, R₂, R₄, R₆, R₈, and R₉ are independently chosen from hydrogen, alkyland aryl; and

R₃, R₅ and R₇ are alkyl groups.

Formula 3 has the structure

wherein

R₁, R₂, R₄, R₆, R₁₀ and R₁₁ are independently chosen from hydrogen,alkyl and aryl; and

R₃, R₅, R₇ and R₉ are alkyl groups.

Formula 4 has the structure

wherein

R₁ and R₅ are independently chosen from methyl, ethyl, n-propyl, andisopropyl;

R₂, R₃, and R₄ are independently chosen from C₁-C₆ alkyl, C₂-C₆ alkenyl,C₃-C₆ cycloalkyl, C₁-C₆ alkyl-C₃-C₆ cycloalkyl-C₁-C₆ alkyl, C₃-C₁₀ aryl,and C₁-C₆ alkyl-C₃-C₁₀ aryl-C₁-C₆ alkyl; and

R₆, R₇, R₈ and R₉ are independently chosen from hydrogen, methyl, andethyl;

Formula 5 has the structure

wherein

R₁ and R₆ are independently chosen from methyl, ethyl, n-propyl, andisopropyl;

R₂, R₃, R₄ and R₅ are independently chosen from C₁-C₆ alkyl, C₂-C₆alkenyl, C₃-C₆ cycloalkyl, C₁-C₆ alkyl-C₃-C₆ cycloalkyl-C₁-C₆ alkyl,C₃-C₁₀ aryl, and C₃-C₁₀ aryl-C₁-C₆ alkyl; and

R₇, R₈, R₉, R₁₀ and R₁₁ are independently chosen from hydrogen, methyl,and ethyl.

In another embodiment, the polyamine analogs are compounds of thestructures 2 and 3, wherein

R₃, R₅, R₇ and R₉ are independently (CH₂)_(x) groups;

x is an integer from 2 to 6; and

R₄, R₆ and R₈ are hydrogen atoms.

In yet another embodiment, the polyamine analogs are compounds of thestructures 2 and 3, wherein

R₃, R₅, R₇ and R₉ are independently (CH₂)_(x) groups;

x is an integer from 2 to 6;

R₄, R₆ and R₈ are hydrogen atoms;

R₁ and R₁₀ are alkyl groups; and

R₂ and R₁₁ are hydrogen atoms.

In yet another embodiment, the polyamine analogs are compounds of thestructures 2 and 3, wherein

R₃, R₅, R₇ and R₉ are independently (CH₂)_(x) groups;

x is an integer from 2 to 6;

R₄, R₆ and R₈ are hydrogen atoms;

R₁ and R₁₀ are alkyl groups;

R₂ and R₁₁ are hydrogen atoms; and

the polyamine analogs have a molecular weight less than 500.

Further embodiments of compounds of the structure 4 include thosewherein R₆, R₇, R₈ and R₉ are hydrogen.

In other embodiments, R₁ and R₅ are ethyl.

In yet further embodiments,

R₆, R₇, R₈ and R₉ are hydrogen; and

R₁ and R₅ are ethyl.

In yet further embodiments,

R₂ and R₄ are independently chosen from C₁-C₆ alkyl; and

R₃ is chosen from C₁-C₆ alkyl, C₂-C₆ alkenyl, C₃-C₆ cycloalkyl, C₁-C₆alkyl-C₃-C₆ cycloalkyl-C₁-C₆ alkyl, C₃-C₁₀ aryl, and C₁-C₆ alkyl-C₃-C₁₀aryl-C₁-C₆ alkyl.

Additional polyamine analogs useful in the present invention includecompounds of the formula 6, and the corresponding stereoisomers, salts,and protected derivatives thereof:

wherein

R₄ is chosen from C₂-C₆ n-alkenyl, C₃-C₆ cycloalkyl, C₃-C₆ cycloalkenyl,and C₃-C₆ aryl;

R₃ and R₅ are independently chosen from a single bond, C₁-C₆ alkyl, andC₁-C₆ alkenyl;

R₂ and R₆ are independently chosen from C₁-C₆ alkyl, C₁-C₆ alkenyl,C₃-C₆ cycloalkyl, C₃-C₆ cycloalkenyl, and C₃-C₆ aryl;

R₁ and R₇ are independently chosen from hydrogen, C₁-C₆ alkyl, and C₂-C₆alkenyl; and

R₈, R₉, R₁₀, and R₁₁ are hydrogen.

In certain embodiments of the compounds of formula 6, R₁ and R₇ areindependently chosen from C₁-C₆ alkyl and C₂-C₆ alkenyl.

Additional polyamine analogs useful in the present invention includecompounds of the formula 7, and the corresponding stereoisomers, salts,and protected derivatives thereof:

wherein

R₄ is chosen from C₁-C₆ n-alkyl and C₁-C₆ branched alkyl;

R₃ and R₅ are independently chosen from a single bond or C₁-C₆ alkyl;

R₂ and R₆ are independently chosen from C₁-C₆ alkyl, C₁-C₆ alkenyl,C₃-C₆ cycloalkyl, C₃-C₆ cycloalkenyl, or C₃-C₆ aryl;

R₁ and R₇ are independently chosen from H, C₁-C₆ alkyl, or C₂-C₆alkenyl; and

R₈, R₉, R₁₀, and R₁₁ are hydrogen.

In certain embodiments of the compounds of formula 7

R₂ and R₇ are independently chosen from C₁-C₆ alkyl or C₂-C₆ alkenyl;

R₄ is chosen from C₁-C₆ saturated n-alkyl and C₁-C₆ saturated branchedalkyl; and

R₃ and R₅ are independently chosen from a single bond and C₁-C₆saturated n-alkyl.

According to another embodiment of the present invention, the agent is achemical moiety that inhibits the activity of S-adenosyl methioninedecarboxylase, inhibits polyamine biosynthesis, and/or increases theactivity of adenosine.

Examples of such moieties include, but are not limited to, those listedin Table 1. Irrespective of the form of the moiety listed in Table 1, itis understood that it includes, as applicable, a salt, protectedderivative, and stereoisomer thereof.

TABLE 1 Pub Chem Compound Official Name (Not IUPAC) ID Decarboxylateds-adenosyl-3-methylthiopropylamine 5351154 SAM Mitoguazone orMethylglyoxal bis(guanylhydrazone) 9561662 “MGBG” EGBG Ethylglyoxalbis(guanylhydrazone) 2354 Berenil Diminazene or Diminazene aceturate4735 Pentamidine 4-[5-(4-carbamimidoylphenoxy)pentoxy]benzenecarboximidamide 5′-(Dimethylsulphino)-5′-deoxyadenosineS-adneosyl-4-methylthiobutyrate S-adenosyl-S-methyl-L-cysteine AMAS-(5′-Deoxy-5′-adenosyl) methylthioethylhydroxylamine EMGBGEthylmethylglyoxal bis(guanylhydrazone) DEGBG Diethylglyoxalbis(guanylhydrazone) 9574151 CGP-33′8296-((2-carbamimidoylhydrazono)methyl) 5479208 picolinimidamide CGP-36′958CGP-39′937 2,2′-bipyridine-6,6′-bis(carboximidamide) CGP-48664 or4-amidinoindan-l-one 2′-amidinohydrazone 5486811 CGP48664Aor SAM 364AAbeAdo 5′-[[(Z)-4-amino-2-butenyl] methylamino]-5′- 6436013 orMDL-73811deoxyadenosine MAOEA 5′-deoxy-5′-[N-methyl-N-[2- 3081018(aminooxy)ethyl]amino] adenosine MHZPA 5′-deoxy-5′-[N-methyl-N-(3-122092 hydrazinopropyl)amino]adenosine MHZEA5′-deoxy-5′-[(2-hydrazinoethyl)- methylamino]adenosine AdoMacS-(5′-deoxy-5′-adenosyl)-1-ammonio-4- 3083364(methylsulfonio)-2cyclopentene AdoMaoS-(5′-deoxy-5′-adenosyl)-1-aminoxy-4- (methylsulfonio)-2-cyclopenteneAPA 1-Aminooxy-3-aminopropane 65020 AOE-PUN-[2-aminooxyethy1]-1,4-diaminobutane AP-APA1-aminooxy-3-N-[3-aminopropyl]-aminopropane 1,11-bis(ethyl)norspermineBES 1,8-bis(ethyl)spermidine BES 1,12-bis(ethyl)spermine DESPMN1,N12-diethylspermine BE-3-3-3 1,11-bis(ethylamino)-4,8-diazaundecanBE-4-4-4 1,14-bis(ethylamino)-5,10-diazatetradecane DEHOP orDiethylhomospermine, N1,N14- DEHSPM diethylhomospermine DENOPdiethyl-norspermine BE-4-4-4-41,19-bis(ethylamino)-5,10,15-triaza-nonadecane SL11037N-ethyl-N′-(2-(3′-ethylamino-propylaminomethyl)-cis-cyclopropylmethyl)-propane 1,3- diamine tetrahydrochlorideSL11038 N-ethyl-N′-(2-(3′-ethylamino-propylaminomethyl)-trans-cyclobutylmethyl)-propane 1,3- diamine tetrahydrochlorideSL11044 N-ethyl-N′-(2-(3′-ethylamino-propylaminomethyl)-transcyclopropylmethyl)-propane 1,3- diamine tetrahydrochlorideSL11047 or N,N′-bis(3-ethylaminopropyl)-cis-but-2-ene-1,4- SL47diaminetetrahydrochloride SL11093 or N,N′-(cyclopropane-1,2- SL93diylbis(methylene))bis(N4-ethylbutane-1,4- diamine)

In yet another embodiment, the agent is a compound chosen from MGBG,MDL73811, CGP48664, Berenil, Pentamidine, SL47, and SL93, or acombination of two or more thereof. In yet another embodiment, the agentis MGBG, SL47 or SL93. In still another embodiment, two or more agentsare used in the methods of the invention to regulate the activity ofosteopontin. The two or more agents can be used either sequentially orsimultaneously.

MGBG is 1,1′[methylethanediylidene]dinitrilodiguanidine and is alsoknown as methylglyoxal bis(guanylhydrazone), methyl-GAG, Me-G, andmitoguazone. As used herein, MGBG includes the free base and saltsthereof. It is commonly, but not necessarily, used as a dihydrochloride.MGBG may be present as any one of the following isomers, or a tautomerand/or a syn/anti isomer thereof, mixture of one or more thereof:

In certain embodiments, MGBG may be present one of the followingisomers, or a tautomer and/or a syn/anti isomer thereof, mixture of oneor more thereof:

In certain embodiments, compounds have a structure chosen from Formulas8a-8c:

R₁-R₆ are chosen from hydrogen, alkyl and aralkyl having from 1 to 12carbon atoms, provided that, in formula (8a), R₁, and R₆ are nothydrogen;

R₇ chosen from hydrogen, alkyl, aryl and aralkyl having from 1 to 12carbon atoms;

m, n, are each independently an integer from 3 to 6, inclusive; and

v, w, x, y, and z are each independently an integer from 3 to 10,inclusive.

Additional disclosure may be found in WO98/10766, the disclosure ofwhich is incorporated by reference as if written herein in its entirety,for example on pp. 3-4.

In certain embodiments, compounds have a structure of Formula 9a:E-NH—B-A-B—NH—B-A-B—NH—B-A-B—NH—B-A-B—NH-E

wherein

A is independently selected from the group consisting of C₁-C₆ alkyl,C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, C₃-C₆ aryl, and C₃-C₆cycloalkenyl;

B is independently selected from the group consisting of: a single bond,C₁-C₆ alkyl, and C₂-C₆ alkenyl; and

E is independently selected from the group consisting of hydrogen, C₁-C₆alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, C₃-C₆ aryl, andC₃-C₆ cycloalkenyl;

with the proviso that either at least one A moiety is selected from thegroup consisting of C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl,C₃-C₆ aryl, and C₃-C₆ cycloalkenyl, or at least one B moiety is selectedfrom the group consisting of C₂-C₆ alkenyl; and all salts, hydrates,solvates, and stereoisomers thereof.

In another embodiment, the conformationally restricted polyamine analogis selected from among the group of compounds of the formula 9b:E-NH—B-A-B—NH—B-A-B—NH—B-A-B—NH(—B-A-B—NH)x-E

wherein:

A is independently selected from the group consisting of C₁-C₆ alkyl,C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, C₃-C₆ aryl, and C₃-C₆cycloalkenyl;

B is independently selected from the group consisting of: a single bond,C₁-C₆ alkyl, and C₂-C₆ alkenyl; and

E is independently selected from the group consisting of hydrogen, C₁-C₆alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, C₃-C₆ aryl, andC₃-C₆ cycloalkenyl;

x is an integer from 2 to 16;

with the proviso that either at least one A moiety is selected from thegroup consisting of C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl,C₃-C₆ aryl, and C₃-C₆ cycloalkenyl, or at least one B moiety is selectedfrom the group consisting of C₂-C₆ alkenyl;

and all salts, hydrates, solvates, and stereoisomers thereof.

In another embodiment, x is 4, 6, 8, or 10.

In another embodiment, x is 4. In another embodiment, x is 6.

In another embodiment, x is 8.

In another embodiment, x is 10.

In another embodiment, the conformationally restricted polyamine analogis selected from among the group of compounds of the formula 9c:E-NH—B-A-B—NH—B-A-B—NH—B-A-B—NH(—B-A-B—NH)x-E

wherein:

A is independently selected from the group consisting of C₁-C₆ alkyl,C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, C₃-C₆ aryl, and C₃-C₆cycloalkenyl;

B is independently selected from the group consisting of: a single bond,C₁-C₆ alkyl, and C₂-C₆ alkenyl; and

E is independently selected from the group consisting of C₁-C₆ alkyl,C₁-C₆ alkanol, C₃-C₆ cycloalkanol, and C₃-C₆ hydroxyaryl, with theproviso that at least one E moiety be selected from the group consistingof C₁-C₆ alkanol, C₃-C₆ cycloalkanol, and C₃-C₆ hydroxyaryl; and

x is an integer from 0 to 16;

and all salts, hydrates, solvates, and stereoisomers thereof.

In another embodiment, the conformationally restricted polyamine analogis selected from among the group of compounds of the formula 9d:E-NH-D-NH—B-A-B—NH-D-NH-E

wherein A is selected from the group consisting of C₂-C₆ alkene andC₃-C₆ cycloalkyl, cycloalkenyl, and aryl;

B is independently selected from the group consisting of a single bondand C₁-C₆ alkyl and alkenyl;

D is independently selected from the group consisting of C₁-C₆ alkyl andalkenyl, and C₃-C₆ cycloalkyl, cycloalkenyl, and aryl;

E is independently selected from the group consisting of hydrogen, C₁-C₆alkyl and alkenyl; and all salts, hydrates, solvates, and stereoisomersthereof.

In another embodiment, the conformationally restricted polyamine analogis selected from macrocyclic polyamines of the formula 9e:

wherein

A₁, each A₂ (if present), and A₃ are independently selected from C₁-C₈alkyl;

each Y is independently selected from hydrogen or C₁-C₄ alkyl;

M is selected from C₁-C₄ alkyl;

k is 0, 1, 2, or 3; and

R is selected from C₁-C₃₂ alkyl;

and all salts, hydrates, solvates, and stereoisomers thereof.

In additional embodiments, the Y group is hydrogen or —CH₃.

In another embodiment, A₁, each A₂ (if present), and A₃ areindependently selected from C₂-C₄ alkyl.

In yet another embodiment, M is —CH2-.

In another embodiment, the conformationally restricted polyamine analogis selected from macrocyclic polyamine analogs of the formula 9f:

wherein

A₁, each A₂ (if present), and A₃ are independently selected from C₁-C₈alkyl;

A₄ is selected from C₁-C₈ alkyl or null;

X is selected from -hydrogen, —Z, —CN, —NH₂, —C(═O)—C₁-C₈-alkyl, or—NHZ, with the proviso that when A₄ is null, X is hydrogen,—C(═O)—C₁-C₈-alkyl, or —Z;

Z is selected from the group consisting of an amino protecting group, anamino capping group, an amino acid, and a peptide;

each Y is independently selected from hydrogen or C₁-C₄ alkyl;

M is selected from C₁-C₄ alkyl;

k is 0, 1, 2, or 3; and

R is selected from C₁-C₃₂ alkyl;

and all salts, hydrates, solvates, and stereoisomers thereof.

In certain embodiments, A₄ is null.

In other embodiments, X is —Z, and —Z is hydrogen.

In other embodiments, X is —Z, and —Z is 4-morpholinocarbonyl.

In other embodiments, X is —Z and —Z is acetyl.

In other embodiments, X is —Z and —Z is t-Boc or Fmoc.

In other embodiments, Y is —CH3.

In other embodiments, M is —CH₂—.

In still further embodiments, k is 1.

In further embodiments, A, and A₃ are —CH₂CH₂CH₂—.

In still further embodiments, —CH₂CH₂CH₂CH₂—.

In still further embodiments, R is C₁₃H₂₇.

In yet further embodiments, one or more of the specific limitations onA₄, X, Z, Y, M, k, A₁, A₃, and R are combined.

In further embodiments of macrocyclic polyamine analog compounds,

-   -   A₄ is C₁-C₈ alkyl;    -   X is —NHZ; and    -   Z is selected from one of the 20 genetically encoded amino acids        (alanine, cysteine, aspartic acid, glutamic acid, phenylalanine,        glycine, histidine, isoleucine, lysine, methionine, asparagine,        proline, glutamine, arginine, serine, threonine, valine,        tryptophan, tyrosine), a peptide of the formula acetyl-SKLQL-, a        peptide of the formula acetyl-SKLQ-I3-alanine-, or a peptide of        the formula acetyl-SKLQ-.

In these cases, where Z is an amino acid or peptide, the therapeuticagent to be used is a polyamine-amino acid conjugate orpolyamine-peptide conjugate.

In one embodiment, the only conformational restriction of the polyamineanalog is due to a carbon-carbon double bond (an ethenyl group, C═C) inthe molecule.

In another embodiment, the only conformational restriction of thepolyamine analog is due to a cycloalkyl group, such as a cyclopropylgroup, in the molecule.

Compounds include, but are not limited to:

Additional disclosure may be found in WO2007/040535, the disclosure ofwhich is incorporated by reference as if written herein in its entirety.

Additional analogs and derivatives include those encompassed by thefollowing formula 10a:R—X-polyaminewherein

R is selected from H or from the group of a straight or branched C1-50saturated or unsaturated aliphatic, carboxyalkyl, carbalkoxyalkyl, oralkoxy; a C1-8 alicyclic; a single or multiring aryl substitutedaliphatic; an aliphatic-substituted single or multiring aromatic; asingle or multiring heterocyclic; a single or multiring heterocyclic 25aliphatic; a C1-10 alkyl; an aryl sulfonyl; or cyano;

X may be —CO—, —SO₂, or —CH₂—, and

“polyamine” may be any naturally occurring, such as putrescine, spermineor spermidine, or synthetically produced polyamine.

Preferably, R is at least about C5, at least about C10, at least aboutC11, at least about C12, at least about C13, at least about C14, atleast about C15, at least about C16, at least about C17, at least aboutC18, at least about C19, at least about C20, or at least about C22.

The linkage between X and the polyamine may be direct, wherein there areno atoms between X and the nitrogen of the amine group of the polyamine,or indirect, where there may be one or more atoms between X and thenitrogen of the amine group of the polyamine. The linkage. between X andthe polyamine may occur via any amino group within the polyamine,although a primary amino group is used in preferred embodiments of theinvention.

In preferred embodiments of the invention where the linkage between Xand the polyamine is indirect, the intervening one or more atoms arepreferably those of an amino acid or a derivative thereof. Inparticularly preferred embodiments of this type, the intervening one ormore atoms are those of lysine, aspartic acid, glutamic acid, ornithine,or 2,4-diaminobutyric acid. Preferred compounds of this type may berepresented as in Formula 10b:R—X-L-polyaminewherein

R is a straight or branched C10-50 saturated or unsaturated aliphatic,carboxyalkyl, carbalkoxyalkyl, or alkoxy; a C1-8 alicyclic; a single ormultiring aryl substituted or unsubstituted aliphatic; analiphatic-substituted or unsubstituted single or multiring aromatic; asingle or multiring heterocyclic; a single or multiring heterocyclicaliphatic; an aryl sulfonyl;

X is —CO—, —SO₂—, or —CH₂—; and

L is a covalent bond or a naturally occurring amino acid, ornithine,2,4-diaminobutyric acid, or derivatives thereof.

The analogs and derivatives of the invention, may be optionally furthersubstituted at one or more other positions of the polyamine. Theseinclude, but are not limited to, internal nitrogen and/or internalcarbon atoms. In one aspect of the invention, preferred substituents arestructures that increase polyamine transport inhibition, bindingaffinity or otherwise enhance the irreversibility of binding of thecompound to a polyamine binding molecule, such as the polyaminetransporter, an enzyme or DNA. Such additional substituents include theaziridine group and various other aliphatic, aromatic, mixedaliphatic-aromatic, or heterocyclic multi-ring structures. Reactivemoieties which, like aziridine, bind covalently to a polyaminetransporter or another polyamine binding molecule, are also within thescope of this invention. Examples of reactive groups that react withnucleophiles to form covalent bonds include chloro-, bromo- andiodo-acetamides, sulfonylfluorides, esters, nitrogen mustards, etc. Suchreactive moieties are used for affinity labeling in a diagnostic orresearch context, and may contribute to pharmacological activity ininhibiting polyamine transport or polyamine synthesis. The reactivegroup can be a reactive photoaffinity group such as an azido orbenzophenone group. Chemical agents for photoaffinity labeling arewell-known in the art (Flemming, S. A., Tetrahedron 1995, 51,12479-12520).

A preferred aspect of the invention relates to a polyamine analog orderivative that is a highly specific polyamine transport inhibitor withpharmaceutical utility as an anticancer chemotherapeutic. One class of apolyamine analog or derivative of the invention that binds to apolyamine-binding site of a molecule and/or inhibits polyaminetransport, is described by the following formula 10c:

wherein

a, b, and c independently range from 1 to 10;

d and e independently range from 0 to 30;

each X is independently either a carbon (C) or sulfur (S) atom, and

R₁ and R₂ are as described below, or each of R₁X(O)n- and of R₂X(O)n-are independently replaced by H; and

* denotes a chiral carbon position;

and with the provisos that

if X is C, then n is 1;

if X is S, then n is 2; and

if X is C, then the X(O) group may be CH₂ such that n is o.

In the above formula, R₁ and R₂ are independently selected from H orfrom the group of a straight or branched C1-50 saturated or unsaturatedaliphatic, carboxyalkyl, carbalkoxyalkyl, or alkoxy; a C1-8 alicyclic; asingle or multiring aryl substituted aliphatic; an aliphatic-substitutedsingle or multiring aromatic; a single or multiring aromatic orsaturated heterocyclic; a single or multiring heterocyclic aliphatic; aC1-10 alkyl; an aryl sulfonyl; or cyano.

Examples of heterocyclic rings as used herein include, but are notlimited to, pyrrole, furan, thiophene, imidazole, oxazole, thiazole,pyrazole, 3-pyrroline, pyrrolidine, pyridine, pyrimidine, purine,quinoline, isoquinoline, and carbazole.

All of the above described aliphatic, carboxyalkyl, carbalkoxyalkyl,alkoxy, 30 alicyclic, aryl, aromatic, and heterocyclic moieties may, ofcourse, also be optionally substituted with 1-3 substituentsindependently selected from halo (fluoro, chloro, bromo or iodo), loweralkyl (1-6C) and lower alkoxy (1-6C).

As used herein, carboxyalkyl refers to the substituent —R′—COOH whereinR′ is alkylene; and carbalkoxyalkyl refers to —R′—COOR wherein R′ and Rare alkylene and alkyl respectively. In preferred embodiments, alkylrefers to a saturated straight- or branched-chain hydrocarbyl radical of1-6 carbon atoms such as methyl, ethyl, n-propyl, isopropyl, n-butyl,t-butyl, n-pentyl, 2-methylpentyl, n-hexyl, and so forth. Alkylene isthe same as alkyl except that the group is divalent. Aryl or alkylsulfonyl moieties have the formula SO₂R and alkoxy moieties have theformula —O—R, wherein R is alkyl, as defined above, or is aryl whereinaryl is phenyl, optionally substituted with 1-3 substituentsindependently selected from halo (fluoro, chloro, bromo or iodo), loweralkyl (1-6C) and lower alkoxy (1-6C).

A preferred group of compounds encompassed by the above is where d is 4and e is 0.

An additional class of a polyamine analog or derivative of the inventionthat binds to a polyamine-binding site of a molecule and/or inhibitspolyamine transport, is described by the following formula 10d:

wherein

a, b, and c independently range from 1 to 10;

d and e independently range from 0 to 30;

R₁ and R₂ are defined as above for formula 8c and

R₃ and R₄ are independently selected from organic substituents including—CH₃ and as defined above for R₁ and R₂ in formula 8c above. Thisgrouping of analogs is produced by reductive amination of the free aminoprecursor with a ketone.

In one preferred embodiment of the invention, R₁ and R₂ are identicaland as described for formula 8c. Positions R₃ and R₄ may also beidentical, and all of R₁ through R₄ may also be identical. Additionally,each of positions R₁, R₂, R₃, and R₄ in formula 8d may also beindependently H.

In an additional aspect of the invention the proximal and/or the distalamino group relative to the polyamine (such as spermine) can bedi-alkylated to form tertiary amines. These materials can be synthesizedby reductive amination with a large excess of the carbonyl component.Additionally, these materials may be produced by a conjugate addition ofthe amine precursor to an α,β-unsaturated carbonyl or α,β-unsaturatednitrile.

Each of R₁, R₂, R₃, and R₄ can be independently varied and are asdefined as above for formula III. Each of R₁, R₂, R₃, and R₄ may also beindependently H. The values of a, b, c, d and e are as described abovefor formula 8d. This aspect of the invention is depicted in thefollowing formula 10e:

In a further aspect of the invention, compounds which lack the proximalor distal amino group on the acyl portion of the molecule are alsoprovided. These are represented by formula 10f:

wherein

-   Z₁ is NR₁R₃ and Z₂ is selected from —R₁, —CHR₁R₂ or —CR₁R₂R₃    (wherein R₁, R₂, and R₃ are as defined above for formula 8c); or Z₂    is NR₂R₄ and Z₁ is selected from —R₁, —CHR₁R₂ or —CR₁R₂R₃ (wherein    R₁, R₂, and R₃ are as defined above for formula 8d). Values for a,    b, and c independently range from 1 to 10; d and e independently    range from 0 to 30. Compounds encompassed by formula V may be    prepared by first coupling amino acid derivatives (modified to    contain the non-amine containing Z group) to a polyamine followed by    appropriate derivatization of the amine containing Z group.    Chemistries for such reactions are known in the art and disclosed    herein.

In preferred embodiments of the invention, positions R₁, R₂, R₃, and R₄,of all the formulas set forth above are independently selected from thefollowing, where each of g, h, i, j, and k are independently selectedfrom 0 to 15:

wherein E refers to “entgegen” and Z refers to “zusammen”.

Compounds include, but are not limited to:

Additional disclosure may be found in WO2002/053519, the disclosure ofwhich is incorporated by reference as if written herein in its entirety.

Additional analogs and derivatives include synthetic derivatives oforiginal polyamines, wherein a carbon atom of said original polyaminecomprises an amide group, said synthetic derivative inhibiting thecellular uptake of a natural polyamine by specifically binding acellular transporter for said natural polyamine.

In certain embodiments, the carbon to which said amido group is locatedis between two internal nitrogen atoms of said original polyamine.

In certain embodiments, the synthetic derivative comprises a dimer ofsaid original polyamine, the monomers of said dimer being linkedtogether by a spacer side chain anchored to the amido group of eachmonomer.

In certain embodiments, the original polyamine is selected from thegroup consisting of putrescine, spermidine and spermine.

In certain embodiments, the original polyamine is spermine.

In certain embodiments, said synthetic derivative has the followinggeneral formula 11a:

in which R₁ and R¹ ₁ independently represent a hydrogen atom or an alkylgroup having 1 to 2 carbon atoms, R₂, R¹ ₂, or R₃ and R¹ ₃ independentlyrepresent a hydrogen atom or a methyl group, wand z independentlyrepresent an integer of 2 or 3, x represents an integer from 0 to n, nrepresents an integer from 3 to 6, the sum of x and y equals n, and Srepresents a hydrogen atom or a molecule which cannot be captured bysaid natural polyamine transporter.

In certain embodiments, said monomer has the following general formula11b:

in which R₁ and R¹ ₁ independently represent a hydrogen atom or an alkylgroup having 1 to 2 carbon atoms, R₂, R¹ ₂, or R₃ and R¹ ₃ independentlyrepresent a hydrogen atom or a methyl group, w and z independentlyrepresent an integer of 2 or 3, x represents an integer from 0 to n, nrepresents an integer from 3 to 6, the sum of x and y equals n, andwherein the spacer side chain comprises a linear hydrocarbon-containingbackbone of 3 to 8 atoms.

In certain embodiments, said backbone comprises sulfur, oxygen ornitrogen.

In certain embodiments, w=2, z=2 x=o and y=3.

In certain embodiments, w=2, z=2, x=o and y=3.

In certain embodiments, w=2, z=2, x=o and y=4.

Compounds include, but are not limited to:

Additional disclosure may be found in WO98/17632, the disclosure ofwhich is incorporated by reference as if written herein in its entirety.

Additional analogs and derivatives include those encompassed by thefollowing formula 12a:

wherein, n can be 0 to 8 and the aminomethyl functionality can be ortho,meta or para substituted, R is hydrogen, —CH₃,—CH₂CH₃, 2-aminoethyl,3-aminopropyl, 4-aminobutyl, 5-aminopentyl, 6-aminohexyl, 7-aminoheptyl,8-aminooctyl, N-methyl-2-aminoethyl, N-methyl-3-aminopropyl,N-methyl-4-aminobutyl, N-methyl-5-aminopentanyl, N-methyl-6-aminohexyl,N-methyl-7-aminoheptyl, N-methyl-8-aminooctyl, N-ethyl-2-aminoethyl,N-ethyl-3-minopropyl, N-ethyl-4-aminobutyl, N-ethyl-5-aminopentyl,N-ethyl-6-aminohexyl, N-ethyl-7-aminoheptyl or N-ethyl-8-aminooctyl andR, is a moiety selected from the group' consisting of a hydrogen or astraight or branched CI-20 saturated or unsaturated aliphatic; aliphaticamine but not propylamine when R═H, n=1 and the aminomethylfunctionality is para substituted; an alicyclic; single or multi-ringaromatic; single or multi-ring aryl substituted aliphatic;aliphatic-substituted single or multi-ring aromatic; a single ormulti-ring heterocyclic, a single or multi-ring heterocyclic-substitutedaliphatic; an aliphatic-substituted aromatic; and halogenated formsthereof.

In certain embodiments, the analogs and derivatives that can be usedaccording to this disclosure can be further modified as described informula 12b:

wherein n can be 0 to 8, R and R₁ are described as above, R₂ can beindependently selected from hydrogen, —CH₃,—CH₂CH₃, and R₃ and R₄ may bethe same or different and are independently selected from hydrogen, orflourine.

In certain embodiments, compounds that can be used according to thisdisclosure are described in formula 12c:

wherein, m and n can be 0 to 7 independently, but m cannot equal n whenR₁ equals R₂ and R₃ equals R₄, o can be 2 to 4, R can be independentlyselected from H, —CH₃,—CH₂CH₃, R₁ and R₂ can be independently selectedfrom hydrogen, —CH₃,—CH₂CH₃, and R₃ and R₄ may be the same or differentand are independently selected from hydrogen or fluorine.

In certain embodiments, compounds have formula 12d:

wherein, R is hydrogen, —CH₃,—CH₂CH₃, m and n can be 0 to 7independently and o

can be 2 to 4, R₂ can be independently selected from hydrogen,—CH₃,—CH₂CH₃, and R₃ and R₄ may be the same or different and areindependently selected from hydrogen or fluorine.

In certain embodiments, compounds of the present invention arerepresented by formula 12e:

wherein, R is hydrogen, —CH₃,—CH₂CH₃, m can be 0 to 7, n can be 0 to 8and o can be 2 to 4, R₂ can be independently selected from hydrogen,—CH₃,—CH₂CH₃, and R₃ and R₄ may be the same or different and areindependently selected from hydrogen or fluorine.

Compounds include, but are not limited to:

Additional disclosure may be found in WO05/105729, the disclosure ofwhich is incorporated by reference as if written herein in its entirety.

Additional analogs and derivatives include compounds of the formulas13a-d:

wherein R₁ and R₂ are independently selected from the group consistingof —C₁-C₁₀ alkyl, —C₃-C₁₀ cycloalkyl, —C₁-C₁₀ alkylene-cycloalkyl,—C₆-C₁₀ aryl, and —C₁-C₁₀ alkylene-aryl, wherein when both R₁ and R₂ arealkyl, at least one of R₁ and R₂ is —C₂-C₁₀ alkyl and wherein both R₁and R₂ are not tert-butyl; and all salts, hydrates, solvates, andstereoisomers thereof; and all mixtures of stereo isomers thereof,including racemic mixtures. In one embodiment, the substituents on thecyclopropyl ring are trans to each other. In another embodiment, thesubstituents on the cyclopropyl ring are cis to each other.

In one embodiment, when both R₁ and R₂ are alkyl, at least one of R₁ andR₂ is straight-chain alkyl. In another embodiment, when both R₁ and R₂are alkyl, both R₁ and R₂ are straight-chain alkyl. In one embodiment,one of R₁ and R₂ is —C₁-C₁₀ alkyl and the other is —C₂-C₁₀ alkyl. In oneembodiment, one of R₁ and R₂ is —C₁-C₁₀, alkyl and the other is —C₄-C₁₀alkyl. In one embodiment, both R₁ and R₂ are —C₄-C₁₀ alkyl. In oneembodiment, one of R₁ and R₂ is —C₆-C₁₀ alkyl. In one embodiment, bothR₁ and R₂ are —C₆-C₁₀ alkyl. In one embodiment, one of R₁ and R₂ is—C₁-C₁₀ alkyl and the other is selected from the group consisting of—C₂-C₄ straight-chain alkyl and —C₄-C₁₀ alkyl. In another embodiment, R₁and R₂ are independently selected from the group consisting of —CH₃,—(CH₂)₃CH₃, and —(CH₂)sCH₃, provided that both R₁ and R₂ are not —CH₃.

In one embodiment, one of R₁ and R₂ is —C₁-C₁₀ alkyl and the other is—C₃-C₁₀ cycloalkyl, —C₁-C₁₀ alkylene-cycloalkyl, —C₆-C₁₀ aryl, or—C₁-C₁₀ alkylene-aryl. In one embodiment, one of R₁ and R₂ is —C₁-C₁₀alkyl and the other is —C₃-C₁₀ cycloalkyl or —C₁-C₁₀alkylene-cycloalkyl. In one embodiment, one of R₁ and R₂ is —C₁-C₁₀alkyl and the other is —C₃-C₁₀ cycloalkyl. In one embodiment, one of R₁and R₂ is —C₁-C₁₀ alkyl and the other is —C₆-C₁₀ aryl or —C₁-C₁₀alkylene-aryl. In one embodiment, one of R₁ and R₂ is —C₁-C₁₀ alkyl andthe other is —C₆-C₁₀ aryl. In another embodiment, both R₁ and R₂ are—C₆-C₁₀ aryl. In another embodiment, both R₁ and R₂ are —C₃-C₁₀cycloalkyl. In one embodiment, the aryl group is benzene. In oneembodiment, the cycloalkyl group is adamantyl. In one embodiment, theadamantyl group is 1-adamantyl. In another embodiment, the adamantylgroup is 2-adamantyl. In another embodiment, R₁ and R₂ are independentlyselected from the group consisting of —CH₃, phenyl, and adamantyl,provided that both R₁ and R₂ are not —CH₃.

Compounds include, but are not limited to:

Additional disclosure may be found in WO2008/112251, the disclosure ofwhich is incorporated by reference as if written herein in its entirety.

Additional analogs and derivatives include compounds of the formula 14a:R₁—X—R₂wherein

R₁ is H, or is a head group selected from the group consisting of astraight or branched C₁₋₁₀ aliphatic, alicyclic, single or multringaromatic, single or multiring aryl substituted aliphatic,aliphatic-substituted single or multiring aromatic, a single ormultiring heterocyclic, a single or multiring heterocyclic-substitutedaliphatic and an aliphatic-substituted aromatic;

R₂ is a polyamine; and

X is CO, NHCO, NHCS, or SO₂

In another embodiment of the above composition, R₂ has the formulaNH(CH₂)_(n)NH(CH₂)_(p)NH(CH₂)_(q)NHR₃wherein

n, p and q vary independently and n=p=q=1 to 12; and

R₃ is H; C₁₋₁₀ alkyl; C₁₋₁₀ alkenyl; C₁₋₁₀ alkynyl; alicyclic; aryl;aryl-substituted alkyl, alkenyl or alkynyl; alkyl-, alkenyl-, oralkynyl-substituted aryl; gauanidino; heterocyclic;heterocyclic-substituted alkyl, alkenyl or alkynyl; and alkyl-,alkenyl-, or alkynyl-substituted heterocyclic.

The above composition may further comprise, linked between X and R₂, alinker L and an additional group Y, such that said composition has theformula 14b:R₁—X-L-Y—R₂wherein

L is a C₁₋₁₀ alkyl; C₁₋₁₀ alkenyl; C₁₋₁₀ alkynyl, alicyclic, orheterocyclic;

X is CO, SO₂, NHCO or NHCS; and

Y is CONH, SO₂NH, NHCO, NHCONH, NHCSNH, NHSO₂, SO₂, O, or S.

In the foregoing compositions R₁ can have the formula:

wherein

R₄, R₅, R₆, R₇, and R₈ are, independently, H, OH, halogen, NO₂,NH₂,NH(CH)_(n)CH₃, N((CH)_(n)CH₃)₂, CN, (CH)_(n)CH₃, O(CH)_(n)CH₃,S(CH2)_(n)CH₃, NCO(CH₂)_(n)CH₃, O(CF₂)_(n)CF₃, or CO—O(CH)_(n)CH₃ wheren=0 to 10.

Alternatively, R₁ has the formula:

wherein

R₄ and R₅ are, independently, H, OH, halogen, NO₂, NH₂,NH(CH)_(n)CH₃,N((CH)_(n)CH₃)₂, CN, (CH)_(n)CH₃, O(CH)_(n)CH₃, S(CH2)_(n)CH₃,NCO(CH₂)_(n)CH₃, O(CF₂)_(n)CF₃, or CO—O(CH)_(n)CH₃ where n=0 to 10.

In yet another embodiment, R₁ has the formula:

wherein

r and s vary independently and r=s=0 to 6;

R₄, R₅, R₆, R₇, R₈, and R₉ are, independently, H, OH, halogen, NO₂,NH₂,NH(CH)_(n)CH₃, N((CH)_(n)CH₃)₂, CN, (CH)_(n)CH₃, O(CH)_(n)CH₃,S(CH2)_(n)CH₃, NCO(CH₂)_(n)CH₃, O(CF₂)_(n)CF₃, or CO—O(CH)_(n)CH₃ wheren=0 to 10;

Q is CONH, SO₂NH, NHCO, NHCONH, NHCSNH, NHSO₂, SO₂, O, or S.

Furthermore, R₁ may have the formula:

wherein

r and s vary independently and are 0 to 6;

R₄, R₅, R₆, and R₇ are, independently, H, OH, NO₂, NH₂,NH(CH)_(n)CH₃,N((CH)_(n)CH₃)₂, CN, (CH)_(n)CH₃, O(CH)_(n)CH₃, S(CH2)_(n)CH₃,NCO(CH₂)_(n)CH₃, O(CF₂)_(n)CF₃, or CO—O(CH)_(n)CH₃ where n=0 to 10; and

Q is CONH, SO₂NH, NHCO, NHCONH, NHCSNH, NHSO₂, SO₂, O, or S.

In the foregoing compositions, R₁ may be selected from the groupconsisting of naphthalene, phenanthrene, anthracene, pyrene,dibenzofuran, acridine, 2,1,3-benzothiodiazole, quinoline, isoquinoline,benzofuran, indole, carbazole, fluorene, 1,3-benzodiazine, phenazine,phenoxazine, phenothiazine, adamantane, camphor, pipiridine,alkylpiperazine, morpholine, cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, cycloheptyl, cyclooctyl, thiophene, furan, pyrrole,alkyl-1,2-diazole, alkylimidazole, alkyl-1H-1,2,3-triazol,alkyl-1H-1,2,3,4-tetrazole, thiazole, oxazole, 1,3,4-thiadiazole,pyridinyl, pyrimidine, 1,2-diazine, 1,4-diazine and 1,3,5-triazine,4-dimethylaminoazobenzene, 3-phenyl-5-methylisooxazole,3-(2-chlorophenyl)-5-methylisooxazole,2-(4-chloropheny)-6-methyl-7-chloroquinoline,6-chloroimidazo[2,1-β]thiazole, α-methylcinnamic acid, and2-[1,2-dihydro-2H-1,4-benzodioxepinyl]thiazole.

R1 may also be a D- or L-amino acid.

Also provided is the above composition where R1 has a formula selectedfrom the group consisting of

wherein

R₁₂ and R₁₃, independently, are H, naphthalene, phenanthrene,anthracene, pyrene, dibenzofuran, acridine, 2,1,3-benzothiodiazole,quinoline, isoquinoline, benzofuran, indole, carbazole, fluorene,1,3-benzodiazine, phenazine, phenoxazine, phenothiazine, adamantane,camphor, pipiridine, alkylpiperazine, morpholine, cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, thiophene,furan, pyrrole, alkyl-I,2-diazole, alkylimidazole,alkyl-1H-1,2,3-triazol, alkyl-1H-1,2,3,4-tetrazole, thiazole, oxazole,1,3,4-thiadiazole, pyridinyl, pyrimidine, 1,2-diazine, 1,4-diazine and1,3,5-triazine, 4-dimethylaminoazobenzene, 3-phenyl-5-methylisooxazole,3-(2-chlorophenyl)-5-methylisooxazole,2-(4-chloropheny)-6-methyl-7-chloroquinoline,6-chloroimidazo[2,1-β]thiazole, α-methylcinnamic acid, or2-[1,2-dihydro-2H-1,4-benzodioxepinyl]thiazole;

and further,

wherein a ring of R₁₂, R₁₃ or both in formulas (A), (B) and (D), isoptionally substituted with one or more of OH, halogen, NO₂,NH₂,NH(CH)_(n)CH₃, N((CH)_(n)CH₃)₂, CN, (CH)_(n)CH₃, O(CH)_(n)CH₃,S(CH2)_(n)CH₃, NCO(CH₂)_(n)CH₃, O(CF₂)_(n)CF₃, or CO—O(CH)_(n)CH₃ wheren=0 to 10

R₁₄ and R₁₅ and, in formula (C), R₁₃, independently, are (CH₂)_(n),(CH₂)_(n)CH═CH, (CH₂)_(n)(CH═CH)_(m)CO, or (CH₂)_(n)CO where n=0 to 5and m=1 to 3;

Y₁, and Z₁, independently, are CONH, SO₂NH, NHCO, NHCONH, NHCSNH, NHSO₂,SO₂—NHSO₂, SO₂, O, S, or COO;

or

when R₁ is of formula (A) or (B), Y1 represents a bond between a C or Natom of R₁₂, and a C or N atom of R₁₃, and Z₁ represents a bond betweena C or N atom of R₁₃, and a C or N atom of R₁₄; or

when R₁ is of formula (C) or Y₁ represents a bond between the C and a Cor N atom of R₁₃ and Z₁ represents a bond between the C and a C or Natom of R₁₄; or

when R₁ is of formula (D) Y₁ represents a bond between a C or N atom ofR₁₂ and a C or N atom of R₁₄ and Z₁ represents a bond between a C or Natom of R₁₃ and a C or N atom of R₁₅.

In the above compositions, R₂ preferably has the formulaNHCH(Z₁)(CH₂)_(n)NH(CH₂)_(p)NH(CH₂)_(q)CH(Z1)NHR₃wherein

n, p and q vary independently and n=p=q=1 to 12; and

R₃ is H; C₁₋₁₀ alkyl; C₁₋₁₀ alkenyl; C₁₋₁₀ alkynyl; alicyclic; aryl;aryl-substituted alkyl, alkenyl or alkynyl; alkyl-, alkenyl-, oralkynyl-substituted aryl; guanidine or heterocyclic; and

Z, is CH₃, CH₂CH₃ or cyclopropyl.

In another embodiment, R₂ has the formula:

wherein

x=1 to 4; y=1 to 3,

R₁₀ and R₁₁ are, independently, H, (CH₂)nNHR₁₂ or (CH₂)_(k)NH(CH₂)1NHR₁₂,

where n=k=1=1 to 10, and R₁₂ is H or C(N═H)NH₂.

In the above compositions, R₂ is preferably selected from the groupconsisting of N1-acetylspermine, N1-acetylspermidine,N8-acetylspermidine, N′-guanidinospermine, cadaverine,aminopropylcadaverine, homo spermidine, caldine(horspermidine),7-hydroxyspermidine, thermine(norspermine), thermospermine, canavalmine,aminopropylhomospermidine, N,N′-bis(3-aminopropyl)cadaverine,aminopentylnorspermidine, N4-aminopropylnorspermidine,N4-aminopropylspermidine, caldopentamine, homocaldopentamine,N4-bis(aminopropyl)norspermidine, thermopentamine,N4-bis(aminopropyl)spermidine, caldohexamine, homothermohexamine,homocaldohexamine, N-(3-aminopropyl)-1,3-propanediamine,N,N′-bis(3-aminopropyl)ethylendiamine,N,N-bis(3-aminopropyl)-1,4-piperazine,N,N-bis(3-aminopropyl)-1,3-piperazine,N,N′-bis(3-aminopropyl)-1,3-propanediamine,N,N′-bis(2-aminoethyl)-1,3-propanediamine, tris(3-aminopropyl)amine, andtris(aminoethyl)amine.

Compounds include, but are not limited to:

Additional disclosure may be found in WO99/03823, the disclosure ofwhich is incorporated by reference as if written herein in its entirety.

Yet further compounds include, but are not limited to:

Additional disclosure may be found in: Ackermann, J M; Pegg, A E;McCloskey, D E; Progress in Cell Cycle Research, 2003, Vol. 5, 461-468;Ekelund, S; Nygren, P; Larsson, R; Biochemical Pharmacology, 2001, 61,1183-1193; Huang, Y; Pledgie, A; Casero Jr, R A; Davidson, N E;Anti-Cancer Drugs, 2005, 16, 229-241; and Marton, J L; Annu. Rev.Pharmacol Toxicol. 1995, 35, 55-91; the disclosure of which isincorporated by reference as if written herein in their entireties.

Polyamine analogs depicted above may be prepared both as salts and asfree bases. In certain embodiments, the salt is the hydrochloride salt.In certain embodiments, the number of coordinated ion pairs (for exampleH⁺C⁻) will be proportional to the number of amino groups in thepolyamine. Such coordination typically occurs at said amino groups,forming, for example, NH₃ ⁺Cl⁻ groups. However, not every amino groupmay be coordinated. For example, if the amino group is adjacent to anelectron-withdrawing group such as carbonyl or sulfonyl, it may notretain sufficient electron density to coordinate an ion. In furtherembodiments, the number of coordinated ions will be proportional to thenumber of primary and/or secondary amino groups in the polyamine.

Additional compounds which may be used in the methods and compositionsdescribed herein include: naturally occurring polyamines found inprokaryotes and eukaryotic cells, polyamine analogs, polyaminebiosynthesis inhibitors, and polyamine transport inhibitors.

Naturally occurring polyamines found in prokaryotes and eukaryotic cellsinclude, but are not limited to: putrescine, spermidine, spermine,diaminopropane, cadaverine, norspermidine, aminopropylcadaverine,homospermine, norspermine, thermospermine, aminopentylnorspermidine,bis(aminopropyl)cadaverine, aminopropylhomospermine, 30 canavalmine,homospermine, caldopentamine, aminopropylcanavaline,bis(aminopropyl)homospermidine, bis(aminobutyl)norspermidine,aminobutylcanavalmine, aminopropylhomospermine, homopentamine,N5-aminobutylhomospermine, caldohexamine, thermohexamine,homothermohexamine, agmatine and N6-methylagmatine. See, e.g., Morgan D.M. L., 1999, Molecular Biotechnology 11: 229.

Polyamine analogs include, but are not limited to, BE-4444[1,19-bis(ethylamino)-5,10,15-triazanonadecane]; BE-3-3-3[N1,N11-diethylnorspermine; DENSPM;1,11-bis(ethylamino)-4,8-diazaundecane; thermine; Warner-Parke-Davis];BE-3-3[N1,N7-bis(ethyl)norspermidine];BE-3-4[N1,N8-bis(ethyl)spermidine]; BE44[N1,N9-bis(ethyl)homospermidine]; BE-343 [N1,N12-bis(ethyl)spermine;diethylspermine-N1-N12; DESPM]; BE-373[N,N′-bis(3-ethylamino)propyl)-1,7-heptane diamine, Merrell-Dow];BE-4-4-4 [N1,N14-bis(ethyl)homospermine; diethylhomospermine-N1-N1-1];BE-3-4-4-3 [1,17-bis(ethylamino)-4,9,14triazaheptadecane]; BE-4-3-3-4[1,17-bis(ethylamino)-5,9,13-triazaheptadecane]; and 1,12-Mez-SPM[1,12-dimethylspermine]. (WOO2007/040535).

Polyamine synthesis inhibitors include but are not limited to:inhibitors of ornithine decarboxylase such as DFMO, aceylenicputrescine, 1-aminooxy-3-aminopropane, antizyme, 2-butylputrescine,cadaverine, L-canaline,5′-deoxy-5′-[N-methyl-N-[3(aminooxy)ethyl]amino]adenosine,5′-deoxy-5′-[N-methyl-N-[3-(hydrazinopropyl)amino]adenosine,diaminopropane, 1,3-diamino-2-propanol, 2-difluoromethyl putrescine,difluorophenylethyl(4-aminopropylamidinohydrazone),2,3-dimethylputrescine, N-dimethylputrescine, 2-ethylputrescine, (+or−)-alphafluoromethylornithine, 2-fluoro methylputrescine,2-hexylputrescine, 2-hydrazinoornithine, ibuprofen, D-methyl acetylenicputrescine, methylglyoxal bis(3aminopropylamininohydrazone),2-methylornithine, 2-methylputrescine,2-monofluoromethyl-trans-dehydorornithine, 2-monofluoromethyldehydroputrescine, monofluoromethylomithine, 2-monofluoromethylputrescine, neomycin, D-omithine, 2-pentylputrescine,p-phenylenediamine, phosphopeptide MG 25000, phosphothreonine,phosphotyrosine, 2-propylputrescine, putrescine,allo-S-adenosyl-L-methionine, S-ethylthioadenosine, methylthioadenosine,and 5′-methyl-thioadenosine as discussed in Zollner H. (1993) Handbookof Enzyme Inhibitors, 2nd Ed. Weinheim:Basel (Switzerland); inhibitorsof S-adenosylmethionine decarboxylase, such as SAM486A(4-aminoindanon-1(2′ amidino)hydrazone dihydrochloride monohydrate),S-adenosyl-1,8-diamino-3thiooctane,S-(5′-adenosyl)methylthio-2-aminooxyethan,S-adenosyl-3-methylthio-i-propylamine,5′-{[(Z)-4-amino-2-butenyl]methylamino}-5′-deoxyadenosine,5′-amino-5′deoxyadenosine,5′-[(aminoiminomethyl)amino]-5′]deoxyadenosine dihydrogensulphate,1-aminooxy-3-aminopropane,[2-(aminooxy)ethyl](5′-deoxyadenosine-5′yl)(methyl)sulphonium,5′-[(3-aminopropyl]-amino)-5′-deoxyadenosine,5′-[(3aminopropyl]-methylamino)-5′-deoxyadenosine,9-[6(RS)-amino-5,6,7-trideoxy-beta-D-ribo-octofuranosyl]-9H-purin-6-amine,borohydride, n-butylglyoxal bis(guanylhydrazone),9-[6(RS)-c-carboxamido-5,6,7-trideoxy-beta-D-ribo-octofuranosyl]-9H-purin-6-amine,cyanide, cyanoborohydride, S-(5′deoxy-5′adenosyl)methionylethylhydroxylamine, S-(5′deoxy-5′adenosyl)methionylthiohydroxylamine,5′-deoxy-5′-[N-methyl-N-[2(aminooxy)ethyl]amino]adenosine,9-[6(S)-diamino-5,6,7,8,9-pentadeoxy-beta-D-ribo-nanofuranosyl]-9H-purin-6-amine,diethylglyoxal bis(guanylhydrazone),difluorophynylethyl(4-aminopropylamidinohydrazone),dimethyl(5′-adenosyljsulfonium, dimethylglyoxal bis(guanylhydrazone),ethylglyoxal bis(guanylhydrazone), hydroxylamine, 4-hydroxypenenal, MDL73811,5′[[3-methylamino)propyl]amino]-5′-deoxyadenosine(1,1′-(methylethanediylidine)dinitro)bis(3-aminoguanididne),methylglyoxal bis(3-aminopropylamidinohydrazone), methylglyoxalbis(cyclohexylamidinohydrazone), pentanedialdehyde bis(guanylhydrazone),phenylhydrazine, propanedialdehyde bis(guanylhydrazone), semicarbazide,sodium borohydride, sodium cyanoborohydride, and spermine as discussedin Zollner H. (1993) Handbook of Enzyme Inhibitors, 2nd Ed. Additionaldisclosure may be found in WO2002/053519, the disclosure of which isincorporated by reference as if written herein in its entirety.

Additional spermine analogs includeN-(2-mercaptoethyl)spermine-5-carboxamide (MESC), the disulfide fromthereof, namely 2,2,1-dithiobis(N-ethyl-spermine-5-carboxamide) (DESC),and N-[2,2,1-dithio(ethyl 1,1-aminoethyl)]spermine-5-carboxamide(DEASC). (WO98/17623)

Polyamine effectors that are small molecule inhibitors or modulators ofkey enzymes in the polyamine biosynthetic pathway include, but are notlimited to: ODC inhibitors such as difluoromethylomithine (DFMO),alpha-monofluoromethylomithine (MFMO), and methyl acetylenicputrescine(MAP); AdometDC inhibitors such asS-(5-deoxy-5adenoxyl)methyIthioethylhydroxylamine (AMA),5-deoxy-5-[(2aminooxyethyl)methyllamino]adenosine (MAOEA), andmethylglyoxal bis(guanylhydrazone) (MGBG); spermidine synthaseinhibitors such as S-adenosyl1,8-diamino-3-thiooctane (AdoDATO),cyclohexylamine, and butylamine; spermine synthase inhibitors such asS-adenosyl-1,12-diamino-3-thio-9-azadodecane (AdoDATAD) andN-(n-butyl)-1,3-diaminopropane (BDAP).

In certain embodiments, the polyamine effector is a polyamine orarginine analog that carries a functional group that confers a cellularor DNA protective effect to the molecule, or that modulates thepolyamine biosynthetic or catabolic pathway. Compounds of this natureinclude, but are not limited to amifostine, NG-hydroxy-arginine (NORA),N1, N11-bis(ethyl)norspermine (BE-3-3-3), N12-bis(ethyl)spermine(BE-3-4-3), N,N-bis[3-(ethylamino)-propyl]-1,7heptanediamine (BE-3-7-3),BE-3-3-3, BE-3-4-3, BE-3-7-3,N1-ethyl-N11-propargyl 4,8-diazaundecane,and the analogs SL-11141 and SL-II050 (structures set forth in one ormore of U.S. Pat. No. 5,889,061, Valasinas et aI., 2001,supra, WO00/66587 and WO 02/38105). Additional disclosure may be found inWO03/013245, the disclosure of which is incorporated by reference as ifwritten herein in its entirety.

As used herein, the terms below have the meanings indicated.

The term “cytokine,” as used herein, alone or in combination, meanssignaling molecules secreted by cells of the immune system which have alocal immunoregulatory effect. Cytokines may include, withoutlimitation, IL-1, IL1-Ra, IL-2, IL-6, IL8, IFNγ, IP-10, IL-17, MCP-1,MMP-9, MIP-1β, TNF-α, TGFβ, CRP, OPN, and RANTES.

The term “osteopontin” is used interchangeably with “OPN,” “SPP1,”“Eta-1,” sialoprotein I or 44K BPP (bone phosphoprotein). In general,osteopontin refers to any full length or partial fragment of afull-length osteopontin. Osteopontin can also refer to any modified,e.g., glycosylated, osteopontin.

The term “activity” as used herein in connection with osteopontin refersto both, the biological activity of the polypeptide and to the quantityor level of osteopontin present in the cell. In one embodiment, the termactivity refers to the quantity of osteopontin, e.g., present, expressedor produced in the cell. In another embodiment, it refers to the levelof osteopontin secreted by the cell, for example, by a mononuclear cell.

When ranges of values are disclosed, and the notation “from n₁ . . . ton₂” or “between n₁ . . . and n₂” is used, where n₁ and n₂ are thenumbers, then unless otherwise specified, this notation is intended toinclude the numbers themselves and the range between them. This rangemay be integral or continuous between and including the end values. Byway of example, the range “from 2 to 6 carbons” is intended to includetwo, three, four, five, and six carbons, since carbons come in integerunits. Compare, by way of example, the range “from 1 to 3 μM(micromolar),” which is intended to include 1 μM, 3 μM, and everythingin between to any number of significant figures (e.g., 1.255 μM, 2.1 μM,2.9999 μM, etc.).

The term “about,” as used herein, is intended to qualify the numericalvalues which it modifies, denoting such a value as variable within amargin of error. When no particular margin of error, such as a standarddeviation to a mean value given in a chart or table of data, is recited,the term “about” should be understood to mean that range which wouldencompass the recited value and the range which would be included byrounding up or down to that figure as well, taking into accountsignificant figures.

The term “substantially” as used herein is intended to meanpredominantly or having the overriding characteristic of, such that anyopposing or detracting characteristics reach a level of insignificance.By way of example, a composition “substantially” free of water might notbe absolutely free of all traces of water, but would be sufficientlyanhydrous that any remaining water would not influence the compositionin any significant way. By way of further example, “substantiallydose-limiting side effects” might be side effects which limited a doseto a level which was below that required for therapeutic efficacy.

The term “disease” as used herein is intended to be generallysynonymous, and is used interchangeably with, the terms “disorder,”“syndrome,” and “condition” (as in medical condition), in that allreflect an abnormal condition of the human or animal body or of one ofits parts that impairs normal functioning, is typically manifested bydistinguishing signs and symptoms, and causes the human or animal tohave a reduced duration or quality of life.

A “proliferative disorder” may be any disorder characterized bydysregulated cellular proliferation. Examples include cancers,psoriasis, and atopic dermatitis.

As used herein, “hyperalgesia” means a heightened sensitivity to pain,and can be considered a type of pain or a measure of pain-relatedbehavior.

As used herein, reference to “treatment” of a patient is intended toinclude prophylaxis. Treatment may also be preemptive in nature, i.e.,it may include prevention of disease. Prevention of a disease mayinvolve complete protection from disease, for example as in the case ofprevention of infection with a pathogen, or may involve prevention ofdisease progression. For example, prevention of a disease may not meancomplete foreclosure of any effect related to the diseases at any level,but instead may mean prevention of the symptoms of a disease to aclinically significant or detectable level. Prevention of diseases mayalso mean prevention of progression of a disease to a later stage of thedisease.

The term “combination therapy” means the administration of two or moretherapeutic agents to treat a therapeutic condition or disorderdescribed in the present disclosure. Such administration encompassesco-administration of these therapeutic agents in a substantiallysimultaneous manner, such as in a single capsule having a fixed ratio ofactive ingredients or in multiple, separate capsules for each activeingredient. In addition, such administration also encompasses use ofeach type of therapeutic agent in a sequential manner. In either case,the treatment regimen will provide beneficial effects of the drugcombination in treating the conditions or disorders described herein.

The term “patient” is generally synonymous with the term “subject” andmeans an animal differing from a disease, disorder, or conditiontreatable in accordance with the methods disclosed herein, including allmammals and humans. Examples of patients include humans, livestock suchas cows, goats, sheep, pigs, and rabbits, and companion animals such asdogs, cats, rabbits, and horses. Preferably, the patient is a human.

An “effective amount” or a “therapeutically effective amount” is aquantity of a compound (e.g., MGBG, a polyamine analog, a polyaminebiosynthesis inhibitor or any agent) that is sufficient to achieve adesired effect in a subject being treated. For instance, this can be theamount necessary to treat a disease, disorder, condition, or adversestate (such as pain or inflammation) or to otherwise measurably alter oralleviate the symptoms, markers, or mechanisms of the disease, disorder,condition, or adverse state. As just one example, an effective amountfor the treatment of pain is an amount sufficient to prevent, delay theonset of, or reduce pain or one or more pain-related symptoms in asubject, as measured by methods known in the art. Similar methods ofassessing response to treatment of a number of diseases are well-know inthe art. The effective amount of a compound of the present invention mayvary depending upon the route of administration and dosage form. Inaddition, specific dosages may be adjusted depending on conditions ofdisease, the age, body weight, general health conditions, sex, and dietof the subject, dose intervals, administration routes, excretion rate,and combinations of agents.

The term “low dose,” in reference to a low dose formulation of a drug ora method of treatment specifically employing a “low dose” of a drug,means a dose which for at least one indication is subtherapeutic, or isa fraction of the dose typically given for at least one indication. Takefor example the case of a drug for the treatment of proliferativedisorders—a low dose formulation for the treatment of, say, chronicpsoriasis, might be a fraction of the dose for the treatment of anaggressive cancer. In this way, the dose for one disease might be anamount which would be subtherapeutic for another disease. Alternatively,for a drug which is therapeutic in different individuals or populationsat different doses, and is available in a range of doses, a low dose maybe simply a dose toward the low end of recognized therapeutic efficacy.Chronic diseases represent an embodiment treatable by low doseformulations and methods. Additionally, a subtherapeutic amount of adrug might be used in combination with one or more other drugs(themselves in either therapeutic or subtherapeutic amounts) to yield acombination formulation or treatment which is potentiated, that is, moreefficacious than the expected effects of the sum of the drugs givenalone. A low dose for the treatment of one indication may be two-fold,three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold,nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold,fifty-fold, may be one hundred-fold less than the therapeutic dose for adifferent indication.

The phrase “therapeutically effective” is intended to qualify the amountof active ingredients used in the treatment of a disease or disorder oron the effecting of a clinical endpoint.

The term “therapeutically acceptable” refers to those compounds (orsalts, prodrugs, tautomers, zwitterionic forms, etc.) which are suitablefor use in contact with the tissues of subjects without undue toxicity,irritation, and allergic response, are commensurate with a reasonablebenefit/risk ratio, and are effective for their intended use.

The term “drug” is used herein interchangeably with “compound” and“agent.”

As used herein, a “polyamine” is any of a group of aliphatic,straight-chain amines derived biosynthetically from amino acids;polyamines are reviewed in Marton et al. (1995) Ann. Rev. Pharm.Toxicol. 35:55-91. By “polyamine” is generally meant anaturally-occurring polyamine or a polyamine which is naturally producedin eukaryotic cells. Examples of polyamines include putrescine,spermidine, spermine and cadaverine.

As used herein, a “polyamine analog” is an organic cation structurallysimilar but non-identical to naturally-occurring polyamines such asspermine and/or spermidine and their precursor, diamine putrescine.Polyamine analogs can be branched or un-branched, or incorporate cyclicmoieties. Polyamines may comprise primary, secondary, tertiary, orquaternary amino groups. In one embodiment, all the nitrogen atoms ofthe polyamine analogs are independently secondary, tertiary, orquaternary amino groups, but are not so limited. Polyamine analogs mayinclude imine, amidine and guanidine groups in place of amine groups.The term “polyamine analog” includes stereoisomers, salts and protectedderivatives of polyamine analogs.

A “stereoisomer” is any optical isomer of a compound, includingenantiomers and diastereomers. Unless otherwise indicated, structuralformulae of compounds are intended to embrace all possiblestereoisomers.

A “salt” or “pharmaceutically acceptable salt” is a compound formed bythe replacement of one or more hydrogen atoms with elements or groups,which is composed of anions and cations, which usually ionizes in water;a salt is formed, for instance, by neutralization of an acid by a base.Examples of salts include, but are not limited to, halide, for example,chloride, bromide, or iodide, nitrate, sulfate, bisulfate, phosphate,acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate,tartrate, pantothenate, bitartrate, ascorbate, succinate, maleate,gentisinate, fumarate, gluconate, glucaronate, saccharate, formate,benzoate, glutamate, methanesulfonate, ethanesulfonate, benzensulfonate,p-toluenesulfonate and pamoate (i.e.,1,1′-methylene-bis-(2-hydroxy-3-naphthoate)) salts.

“Protected derivative” is used to refer to a compound protected with aprotecting group. “Protecting group” refers to a chemical group thatexhibits the following characteristics: 1) reacts selectively with thedesired functionality in good yield (preferably at least 80%, morepreferably at least 90%, more preferably at least 95%, still morepreferably at least 99%) to give a protected substrate that is stable tothe projected reactions for which protection is desired; 2) isselectively removable from the protected substrate to yield the desiredfunctionality; and 3) is removable in good yield (preferably at least80%, more preferably at least 90%, more preferably at least 95%, stillmore preferably at least 99%) by reagents compatible with the otherfunctional group(s) present or generated in such projected reactions.Examples of suitable protecting groups can be found in Greene et al.(1991) Protective Groups in Organic Synthesis, 2nd Ed. (John Wiley &Sons, Inc., New York). Exemplary protecting groups for the aminofunctionality include, but are not limited to, mesitylenesulfonyl(MesS02), benzyloxycarbonyl (CBz), t-butyloxycarbonyl (Boc),t-butyldimethylsilyl (TBDIMS), 9-fluorenylmethyloxycarbonyl (Fmoc), orsuitable photolabile protecting groups such as 6-nitroveratryloxycarbonyl (Nvoc).

The term “acyl,” as used herein, alone or in combination, refers to acarbonyl attached to an alkenyl, alkyl, aryl, cycloalkyl, heteroaryl,heterocycle, or any other moiety were the atom attached to the carbonylis carbon. An “acetyl” group refers to a —C(O)CH₃ group. An“alkylcarbonyl” or “alkanoyl” group refers to an alkyl group attached tothe parent molecular moiety through a carbonyl group. Examples of suchgroups include methylcarbonyl and ethylcarbonyl. Examples of acyl groupsinclude formyl, alkanoyl and aroyl.

The term “alkenyl,” as used herein, alone or in combination, refers to astraight-chain or branched-chain hydrocarbon radical having one or moredouble bonds and containing from 2 to 20 carbon atoms. In certainembodiments, said alkenyl will comprise from 2 to 6 carbon atoms. Theterm “alkenylene” refers to a carbon-carbon double bond system attachedat two or more positions such as ethenylene[(—CH═CH—),(—C::C—)].Examples of suitable alkenyl radicals include ethenyl, propenyl,2-methylpropenyl, 1,4-butadienyl and the like. Unless otherwisespecified, the term “alkenyl” may include “alkenylene” groups.

The term “alkoxy,” as used herein, alone or in combination, refers to analkyl ether radical, wherein the term alkyl is as defined below.Examples of suitable alkyl ether radicals include methoxy, ethoxy,n-propoxy, isopropoxy, n-butoxy, iso-butoxy, sec-butoxy, tert-butoxy,and the like.

The term “alkyl,” as used herein, alone or in combination, refers to astraight-chain or branched-chain alkyl radical containing from 1 to 20carbon atoms. In certain embodiments, said alkyl will comprise from 1 to10 carbon atoms. In further embodiments, said alkyl will comprise from 1to 6 carbon atoms. Alkyl groups may be optionally substituted as definedherein. Examples of alkyl radicals include methyl, ethyl, n-propyl,isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, iso-amyl,hexyl, octyl, nonyl and the like. The term “alkylene,” as used herein,alone or in combination, refers to a saturated aliphatic group derivedfrom a straight or branched chain saturated hydrocarbon attached at twoor more positions, such as methylene (—CH₂—). Unless otherwisespecified, the term “alkyl” may include “alkylene” groups.

The term “alkylamino,” as used herein, alone or in combination, refersto an alkyl group attached to the parent molecular moiety through anamino group. Suitable alkylamino groups may be mono- or dialkylated,forming groups such as, for example, N-methylamino, N-ethylamino,N,N-dimethylamino, N,N-ethylmethylamino and the like.

The term “alkynyl,” as used herein, alone or in combination, refers to astraight-chain or branched chain hydrocarbon radical having one or moretriple bonds and containing from 2 to 20 carbon atoms. In certainembodiments, said alkynyl comprises from 2 to 6 carbon atoms. In furtherembodiments, said alkynyl comprises from 2 to 4 carbon atoms. The term“alkynylene” refers to a carbon-carbon triple bond attached at twopositions such as ethynylene (—C:::C—, —C≡C—). Examples of alkynylradicals include ethynyl, propynyl, hydroxypropynyl, butyn-1-yl,butyn-2-yl, pentyn-1-yl, 3-methylbutyn-1-yl, hexyn-2-yl, and the like.Unless otherwise specified, the term “alkynyl” may include “alkynylene”groups.

The terms “amido” and “carbamoyl,” as used herein, alone or incombination, refer to an amino group as described below attached to theparent molecular moiety through a carbonyl group, or vice versa. Theterm “C-amido” as used herein, alone or in combination, refers to a—C(O)N(RR′) group with R and R′ as defined herein or as defined by thespecifically enumerated “R” groups designated. The term “N-amido” asused herein, alone or in combination, refers to a RC(O)N(R′)— group,with R and R′ as defined herein or as defined by the specificallyenumerated “R” groups designated. The term “acylamino” as used herein,alone or in combination, embraces an acyl group attached to the parentmoiety through an amino group. An example of an “acylamino” group isacetylamino (CH₃C(O)NH—).

The term “amino,” as used herein, alone or in combination, refers to—NRR′, wherein R and R′ are independently selected from the groupconsisting of hydrogen, alkyl, acyl, heteroalkyl, aryl, cycloalkyl,heteroaryl, and heterocycloalkyl, any of which may themselves beoptionally substituted. Additionally, R and R′ may combine to formheterocycloalkyl, either of which may be optionally substituted.

The term “aryl,” as used herein, alone or in combination, means acarbocyclic aromatic system containing one, two or three rings whereinsuch polycyclic ring systems are fused together. The term “aryl”embraces aromatic groups such as phenyl, naphthyl, anthracenyl, andphenanthryl.

The term “arylalkyl” or “aralkyl,” as used herein, alone or incombination, refers to an aryl group attached to the parent molecularmoiety through an alkyl group. The term “carboxyl” or “carboxy,” as usedherein, refers to —C(O)OH or the corresponding “carboxylate” anion, suchas is in a carboxylic acid salt. An “O-carboxy” group refers to aRC(O)O— group, where R is as defined herein. A “C-carboxy” group refersto a —C(O)OR groups where R is as defined herein.

The term “cyano,” as used herein, alone or in combination, refers to—CN.

The term “cycloalkyl,” or, alternatively, “carbocycle” or “alicyclic,”as used herein, alone or in combination, refers to a saturated orpartially saturated monocyclic, bicyclic or tricyclic alkyl groupwherein each cyclic moiety contains from 3 to 12 carbon atom ringmembers and which may optionally be a benzo fused ring system which isoptionally substituted as defined herein. In certain embodiments, saidcycloalkyl will comprise from 5 to 7 carbon atoms. Examples of suchcycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, cycloheptyl, tetrahydronapthyl, indanyl, octahydronaphthyl,2,3-dihydro-1H-indenyl, adamantyl and the like. “Bicyclic” and“tricyclic” as used herein are intended to include both fused ringsystems, such as decahydronaphthalene, octahydronaphthalene as well asthe multicyclic (multicentered) saturated or partially unsaturated type.The latter type of isomer is exemplified in general by,bicyclo[1,1,1]pentane, camphor, adamantane, and bicyclo[3,2,1]octane.

The term “halo,” or “halogen,” as used herein, alone or in combination,refers to fluorine, chlorine, bromine, or iodine.

The term “heteroalkyl,” as used herein, alone or in combination, refersto a stable straight or branched chain, or cyclic hydrocarbon radical,or combinations thereof, fully saturated or containing from 1 to 3degrees of unsaturation, consisting of the stated number of carbon atomsand from one to three heteroatoms selected from the group consisting ofO, N, and S, and wherein the nitrogen and sulfur atoms may optionally beoxidized and the nitrogen heteroatom may optionally be quaternized. Theheteroatom(s) O, N and S may be placed at any interior position of theheteroalkyl group. Up to two heteroatoms may be consecutive, such as,for example, —CH₂—NH—OCH₃.

The term “heteroaryl,” as used herein, alone or in combination, refersto a 3- to 15-membered unsaturated heteromonocyclic ring, or a fusedmonocyclic, bicyclic, or tricyclic ring system in which at least one ofthe fused rings is aromatic, which contains at least one atom selectedfrom the group consisting of O, S, and N. In certain embodiments, saidheteroaryl will comprise from 5 to 7 carbon atoms. The term alsoembraces fused polycyclic groups wherein heterocyclic rings are fusedwith aryl rings, wherein heteroaryl rings are fused with otherheteroaryl rings, wherein heteroaryl rings are fused withheterocycloalkyl rings, or wherein heteroaryl rings are fused withcycloalkyl rings. Examples of heteroaryl groups include pyrrolyl,pyrrolinyl, imidazolyl, pyrazolyl, pyridyl, pyrimidinyl, pyrazinyl,pyridazinyl, triazolyl, pyranyl, furyl, thienyl, oxazolyl, isoxazolyl,oxadiazolyl, thiazolyl, thiadiazolyl, isothiazolyl, indolyl, isoindolyl,indolizinyl, benzimidazolyl, quinolyl, isoquinolyl, quinoxalinyl,quinazolinyl, indazolyl, benzotriazolyl, benzodioxolyl, benzopyranyl,benzoxazolyl, benzoxadiazolyl, benzothiazolyl, benzothiadiazolyl,benzofuryl, benzothienyl, chromonyl, coumarinyl, benzopyranyl,tetrahydroquinolinyl, tetrazolopyridazinyl, tetrahydroisoquinolinyl,thienopyridinyl, furopyridinyl, pyrrolopyridinyl and the like. Exemplarytricyclic heterocyclic groups include carbazolyl, benzidolyl,phenanthrolinyl, dibenzofuranyl, acridinyl, phenanthridinyl, xanthenyland the like.

The terms “heterocycloalkyl” and, interchangeably, “heterocycle,” asused herein, alone or in combination, each refer to a saturated,partially unsaturated, or fully unsaturated monocyclic, bicyclic, ortricyclic heterocyclic group containing at least one heteroatom as aring member, wherein each said heteroatom may be independently selectedfrom the group consisting of nitrogen, oxygen, and sulfur In certainembodiments, said hetercycloalkyl will comprise from 1 to 4 heteroatomsas ring members. In further embodiments, said hetercycloalkyl willcomprise from 1 to 2 heteroatoms as ring members. In certainembodiments, said hetercycloalkyl will comprise from 3 to 8 ring membersin each ring. In further embodiments, said hetercycloalkyl will comprisefrom 3 to 7 ring members in each ring. In yet further embodiments, saidhetercycloalkyl will comprise from 5 to 6 ring members in each ring.“Heterocycloalkyl” and “heterocycle” are intended to include sulfones,sulfoxides, N-oxides of tertiary nitrogen ring members, and carbocyclicfused and benzo fused ring systems; additionally, both terms alsoinclude systems where a heterocycle ring is fused to an aryl group, asdefined herein, or an additional heterocycle group. Examples ofheterocycle groups include aziridinyl, azetidinyl, 1,3-benzodioxolyl,dihydroisoindolyl, dihydroisoquinolinyl, dihydrocinnolinyl,dihydrobenzodioxinyl, dihydro[1,3]oxazolo[4,5-b]pyridinyl,benzothiazolyl, dihydroindolyl, dihy-dropyridinyl, 1,3-dioxanyl,1,4-dioxanyl, 1,3-dioxolanyl, isoindolinyl, morpholinyl, piperazinyl,pyrrolidinyl, tetrahydropyridinyl, piperidinyl, thiomorpholinyl, and thelike. The heterocycle groups may be optionally substituted unlessspecifically prohibited.

The term “lower,” as used herein, alone or in a combination, where nototherwise specifically defined, means containing from 1 to and including6 carbon atoms.

The term “sulfonyl,” as used herein, alone or in combination, refers to—S(O)₂—.

Any definition herein may be used in combination with any otherdefinition to describe a composite structural group. By convention, thetrailing element of any such definition is that which attaches to theparent moiety. For example, the composite group alkylamido wouldrepresent an alkyl group attached to the parent molecule through anamido group, and the term alkoxyalkyl would represent an alkoxy groupattached to the parent molecule through an alkyl group.

When a group is defined to be “null,” what is meant is that said groupis absent.

The term “optionally substituted” means the anteceding group may besubstituted or unsubstituted. When substituted, the substituents of an“optionally substituted” group may include, without limitation, one ormore substituents independently selected from the following groups or aparticular designated set of groups, alone or in combination: loweralkyl, lower alkenyl, lower alkynyl, lower alkanoyl, lower heteroalkyl,lower heterocycloalkyl, lower haloalkyl, lower haloalkenyl, lowerhaloalkynyl, lower perhaloalkyl, lower perhaloalkoxy, lower cycloalkyl,phenyl, aryl, aryloxy, lower alkoxy, lower haloalkoxy, oxo, loweracyloxy, carbonyl, carboxyl, lower alkylcarbonyl, lower carboxyester,lower carboxamido, cyano, hydrogen, halogen, hydroxy, amino, loweralkylamino, arylamino, amido, nitro, thiol, lower alkylthio, lowerhaloalkylthio, lower perhaloalkylthio, arylthio, sulfonate, sulfonicacid, trisubstituted silyl, N₃, SH, SCH₃, C(O)CH₃, CO₂CH₃, CO₂H,pyridinyl, thiophene, furanyl, lower carbamate, and lower urea. Twosubstituents may be joined together to form a fused five-, six-, orseven-membered carbocyclic or heterocyclic ring consisting of zero tothree heteroatoms, for example forming methylenedioxy or ethylenedioxy.An optionally substituted group may be unsubstituted (e.g., —CH₂CH₃),fully substituted (e.g., —CF₂CF₃), monosubstituted (e.g., —CH₂CH₂F) orsubstituted at a level anywhere in-between fully substituted andmonosubstituted (e.g., —CH₂CF₃). Where substituents are recited withoutqualification as to substitution, both substituted and unsubstitutedforms are encompassed. Where a substituent is qualified as“substituted,” the substituted form is specifically intended.Additionally, different sets of optional substituents to a particularmoiety may be defined as needed; in these cases, the optionalsubstitution will be as defined, often immediately following the phrase,“optionally substituted with.”

The term R or the term R′, appearing by itself and without a numberdesignation, unless otherwise defined, refers to a moiety selected fromthe group consisting of hydrogen, alkyl, cycloalkyl, heteroalkyl, aryl,heteroaryl and heterocycloalkyl, any of which may be optionallysubstituted. Such R and R′ groups should be understood to be optionallysubstituted as defined herein. Whether an R group has a numberdesignation or not, every R group, including R, R′ and R^(n) where n=(1,2, 3, . . . n), every substituent, and every term should be understoodto be independent of every other in terms of selection from a group.Should any variable, substituent, or term (e.g. aryl, heterocycle, R,etc.) occur more than one time in a formula or generic structure, itsdefinition at each occurrence is independent of the definition at everyother occurrence. Those of skill in the art will further recognize thatcertain groups may be attached to a parent molecule or may occupy aposition in a chain of elements from either end as written. Thus, by wayof example only, an unsymmetrical group such as —C(O)N(R)— may beattached to the parent moiety at either the carbon or the nitrogen.

Asymmetric centers exist in the compounds disclosed herein. Thesecenters are designated by the symbols “R” or “S,” depending on theconfiguration of substituents around the chiral carbon atom. It shouldbe understood that the invention encompasses all stereochemical isomericforms, including diastereomeric, enantiomeric, and epimeric forms, aswell as d-isomers and 1-isomers, and mixtures thereof. Individualstereoisomers of compounds can be prepared synthetically fromcommercially available starting materials which contain chiral centersor by preparation of mixtures of enantiomeric products followed byseparation such as conversion to a mixture of diastereomers followed byseparation or recrystallization, chromatographic techniques, directseparation of enantiomers on chiral chromatographic columns, or anyother appropriate method known in the art. Starting compounds ofparticular stereochemistry are either commercially available or can bemade and resolved by techniques known in the art. Additionally, thecompounds disclosed herein may exist as geometric isomers. The presentinvention includes all cis, trans, syn, anti, entgegen (E), and zusammen(Z) isomers as well as the appropriate mixtures thereof. Additionally,compounds may exist as tautomers; all tautomeric isomers are provided bythis invention. Additionally, the compounds disclosed herein can existin unsolvated as well as solvated forms with pharmaceutically acceptablesolvents such as water, ethanol, and the like. In general, the solvatedforms are considered equivalent to the unsolvated forms.

The term “bond” refers to a covalent linkage between two atoms, or twomoieties when the atoms joined by the bond are considered to be part oflarger substructure. A bond may be single, double, or triple unlessotherwise specified. A dashed line between two atoms in a drawing of amolecule indicates that an additional bond may be present or absent atthat position.

A “macrophage” is a phagocytic cell, some are fixed and others circulatein the blood stream. Macrophages are regulatory and effector cell of theimmune response. These cells are susceptible to infection by viruses. Asused herein, the terms “macrophage” and “monocyte” are usedinterchangeably, as it is understood that in the art the term “monocyte”is often used to describe a circulating mononuclear cell that expressesthe CD14 cell surface marker, and when in a tissue this cell is alsoclassified as a macrophage.

A “macrophage-associated condition” is a condition, disorder, orindication that is associated with an elevated or abnormal level ofmacrophage proliferation or activation as compared to control sample(s).Such disorders include, but are not limited to, AIDS-associateddementia, Alzheimer's disease (AD), Amyotrophic Lateral Sclerosis (ALS)AIDS lymphoma, follicular lymphoma, mycoses fungoides, T cell and B celllymphomas with significant macrophage compartments, age related maculardegeneration (ARMD), wet and dry forms, atherosclerosis, kidney diseasesuch as focal segmental glomerulosclerosis, and membrane proliferativeglomerulonephropathy, lupus, psoriaform dermatitis, AIDS-associateddiarrhea, prelymphomatic autoimmune disease such as AILD(angioimmunoblastic lymphadenopathy with dysproteinemia), chronichepatitis viral diseases (HBV and HCV), peripheral sensory neuropathyassociated with HIV infection or diabetes mellitus and herpes virusassociated diseases such as Castleman's disease and Kaposi's sarcoma. Inone embodiment, they include invasive breast cancer and pancreaticcancer. The terms “condition,” “disorder,” and “disease” are usedinterchangeably herein. “Macrophage-associated dementia” is a dementiathat is associated with an elevated, or abnormal, level of macrophageproliferation or activation as compared to control sample(s). Suchdementias include, but are not limited to, AD. A macrophage-associateddisorder, disease or dementia can be HIV-mediated or non-HIV-mediated,or HIV-associated or non-HIV associated. A “non-HIV-mediated” disease ordementia is a disease or dementia which is not caused by HIV, eitherdirectly or indirectly. A “non-HIV-associated” disease or dementia isnot normally associated with or secondary to HIV infection. An“HIV-mediated” disease, dementia, or indication is directly orindirectly caused by (and/or linked to) HIV infection. An“HIV-associated” disease, dementia or indication is defined more broadlyas generally associated with or secondary to an HIV infection;“HIV-mediated” diseases, for example, are included in those consideredto be “HIV-associated.” The terms “HIV neuropathy” and “HIV-associatedneurodegeneration” (“HAND”) may be used herein interchangeably.HIV-associated dementia may also be used interchangeably with HAD, HIVdementia, AIDS dementia, AIDS dementia complex, ADC, and neuro AIDS.

A “virus” is a microscopic infectious organism that reproduces insideliving cells. A virus consists essentially of a core of a single nucleicacid surrounded by a protein coat, and has the ability to replicateinside a living cell. The term virus includes retroviruses, which areRNA viruses wherein the viral genome is RNA, and lentiviruses, aclassification which describes a genus of viruses containing reversetranscriptase.

HIV is a retrovirus that causes immunosuppression in humans (HIVdisease), and leads to a disease complex known as the acquiredimmunodeficiency syndrome (AIDS). “HIV disease” refers to awell-recognized group of signs and symptoms in persons infected by anHIV virus.

“Viral load” is a measure of the severity of a viral infection, and canbe estimated by calculating the amount of virus in a body fluid or ininfected cells. Viral load may be employed as a surrogate marker fordisease progression. Viral load is typically measured by PCR and bDNAtests and is generally expressed in number of virus copies orequivalents per milliliter. For example, “HIV viral load” may bemeasured by determining the level of HIV-RNA (measured in copies per ml)detectable by polymerase chain reaction (PCR) in the plasma of anHIV-infected subject.

A “clinically significant” reduction in HIV viral load includes areduction greater than or equal to about 80% (a half log) relative to abaseline value. Similarly, a “clinically significant” reduction in thenumber of HIV-infected CD14/CD16+ blood macrophages includes a reductionof at least about 80% relative to a baseline value.

The term “prodrug” refers to a compound that is made more active invivo. Certain compounds disclosed herein may also exist as prodrugs, asdescribed in Hydrolysis in Drug and Prodrug Metabolism: Chemistry,Biochemistry, and Enzymology (Testa, Bernard and Mayer, Joachim M.Wiley-V H C A, Zurich, Switzerland 2003). Prodrugs of the compoundsdescribed herein are structurally modified forms of the compound thatreadily undergo chemical changes under physiological conditions toprovide the compound. Additionally, prodrugs can be converted to thecompound by chemical or biochemical methods in an ex vivo environment.For example, prodrugs can be slowly converted to a compound when placedin a transdermal patch reservoir with a suitable enzyme or chemicalreagent. Prodrugs are often useful because, in some situations, they maybe easier to administer than the compound, or parent drug. They may, forinstance, be bioavailable by oral administration whereas the parent drugis not. The prodrug may also have improved solubility in pharmaceuticalcompositions over the parent drug. A wide variety of prodrug derivativesare known in the art, such as those that rely on hydrolytic cleavage oroxidative activation of the prodrug. An example, without limitation, ofa prodrug would be a compound which is administered as an ester (the“prodrug”), but then is metabolically hydrolyzed to the carboxylic acid,the active entity. Additional examples include peptidyl derivatives of acompound.

The compounds disclosed herein can exist as therapeutically acceptablesalts. The present invention includes compounds listed above in the formof salts, including acid addition salts. Suitable salts include thoseformed with both organic and inorganic acids. Such acid addition saltswill normally be pharmaceutically acceptable. However, salts ofnon-pharmaceutically acceptable salts may be of utility in thepreparation and purification of the compound in question. Basic additionsalts may also be formed and be pharmaceutically acceptable. For a morecomplete discussion of the preparation and selection of salts, refer toPharmaceutical Salts: Properties, Selection, and Use (Stahl, P.Heinrich. Wiley-V C H A, Zurich, Switzerland, 2002).

The term “therapeutically acceptable salt,” as used herein, representssalts or zwitterionic forms of the compounds disclosed herein which arewater or oil-soluble or dispersible and therapeutically acceptable asdefined herein. The salts can be prepared during the final isolation andpurification of the compounds or separately by reacting the appropriatecompound in the form of the free base with a suitable acid.Representative acid addition salts include acetate, adipate, alginate,L-ascorbate, aspartate, benzoate, benzenesulfonate (besylate),bisulfate, butyrate, camphorate, camphorsulfonate, citrate, digluconate,formate, fumarate, gentisate, glutarate, glycerophosphate, glycolate,hemisulfate, heptanoate, hexanoate, hippurate, hydrochloride,hydrobromide, hydroiodide, 2-hydroxyethansulfonate(isethionate),lactate, maleate, malonate, DL-mandelate, mesitylenesulfonate,methanesulfonate, naphthylenesulfonate, nicotinate,2-naphthalenesulfonate, oxalate, pamoate, pectinate, persulfate,3-phenylproprionate, phosphonate, picrate, pivalate, propionate,pyroglutamate, succinate, sulfonate, tartrate, L-tartrate,trichloroacetate, trifluoroacetate, phosphate, glutamate, bicarbonate,para-toluenesulfonate (p-tosylate), and undecanoate. Also, basic groupsin the compounds disclosed herein can be quaternized with methyl, ethyl,propyl, and butyl chlorides, bromides, and iodides; dimethyl, diethyl,dibutyl, and diamyl sulfates; decyl, lauryl, myristyl, and sterylchlorides, bromides, and iodides; and benzyl and phenethyl bromides.Examples of acids which can be employed to form therapeuticallyacceptable addition salts include inorganic acids such as hydrochloric,hydrobromic, sulfuric, and phosphoric, and organic acids such as oxalic,maleic, succinic, and citric. Salts can also be formed by coordinationof the compounds with an alkali metal or alkaline earth ion. Hence, thepresent invention contemplates sodium, potassium, magnesium, and calciumsalts of the compounds disclosed herein, and the like.

Basic addition salts can be prepared during the final isolation andpurification of the compounds by reacting a carboxy group with asuitable base such as the hydroxide, carbonate, or bicarbonate of ametal cation or with ammonia or an organic primary, secondary, ortertiary amine. The cations of therapeutically acceptable salts includelithium, sodium, potassium, calcium, magnesium, and aluminum, as well asnontoxic quaternary amine cations such as ammonium, tetramethylammonium,tetraethylammonium, methylamine, dimethylamine, trimethylamine,triethylamine, diethylamine, ethylamine, tributylamine, pyridine,N,N-dimethylaniline, N-methylpiperidine, N-methylmorpholine,dicyclohexylamine, procaine, dibenzylamine, N,N-dibenzylphenethylamine,1-ephenamine, and N,N-dibenzylethylenediamine. Other representativeorganic amines useful for the formation of base addition salts includeethylenediamine, ethanolamine, diethanolamine, piperidine, andpiperazine.

While it may be possible for the compounds disclosed herein to beadministered as the raw chemical, it is also possible to present them asa pharmaceutical formulation. Accordingly, provided herein arepharmaceutical formulations which comprise one or more of certaincompounds disclosed herein, or one or more pharmaceutically acceptablesalts, esters, prodrugs, amides, or solvates thereof, together with oneor more pharmaceutically acceptable carriers thereof and optionally oneor more other therapeutic ingredients. The carrier(s) must be“acceptable” in the sense of being compatible with the other ingredientsof the formulation and not deleterious to the recipient thereof. Properformulation is dependent upon the route of administration chosen. Any ofthe well-known techniques, carriers, and excipients may be used assuitable and as understood in the art; e.g., in Remington'sPharmaceutical Sciences. The pharmaceutical compositions disclosedherein may be manufactured in any manner known in the art, e.g., bymeans of conventional mixing, dissolving, granulating, dragee-making,levigating, emulsifying, encapsulating, entrapping or compressionprocesses.

The agent—a polyamine analog, polyamine biosynthesis inhibitor,polyamine transport inhibitor, or agent that inhibits S-adenosylmethionine decarboxylase—may also be administered in combination withone or more entities. In one embodiment, the entity is a therapeuticentity, including, but not limited to, an anti-viral or anti-retroviralagent, a steroid or other anti-inflammatory agent. In anotherembodiment, the entity is a pharmaceutically acceptable carrier.

The effective amount of an agent that inhibits S-adenosyl methioninedecarboxylase, e.g., in a cell or a subject, can be any amount that issufficient to decrease the level or activity of osteopontin, e.g., inthe cell or the subject, typically by about 25%, 30%, 40%, 50%, 60%,70%, 75%, 80%, 85%, 90%, 95% or more. In one embodiment, the effectiveamount of an agent is an amount that is sufficient to decrease theactivity of osteopontin by 70% or more. In another embodiment, theeffective amount of an agent is an amount that is sufficient to decreasethe activity of osteopontin by 80% or more. In yet another embodiment,the agent inhibits S-adenosyl methionine decarboxylase and the effectiveamount is an amount sufficient to activate adenosine deaminase (“ADA”).

The optimal dose, frequency of administration, and duration of treatmentwith the agent in a subject may vary from subject to subject, dependingon the disease to be treated or clinical endpoint to be reached (forexample, decrease in the level or activity of osteopontin, inhibition ofinfiltration of macrophages to a tissue, or mitigation of pain) thesubject's condition, the subject's age, weight, response to thetreatment, and the nature of the therapeutic entity. Determination ofthe optimal dose and duration of treatment is within the scope of one ofskill in the art. The optimal dose and duration of treatment may be bestdetermined by monitoring the subject's response during the course of thetreatment. In some instances, the administration of higher doses maypermit less frequent administration, and lower doses may require morefrequent administration in order to achieve a clinically significantimprovement in the subject's condition. The agent(s) of the inventionmay be administered as a single dose or in multiple doses.

Generally, a therapeutically effective dose of the agent in accordancewith the present methods will be one or more doses of from about 10 toabout 1100 mg/m². Lower dose regimens include doses of 10-200, 10-100,10-50 and 20-200 mg/m². Higher dose regimens include 200-400, 250-500,400-600, 500-800 600-1000 and 800-1100 mg/m². In one embodiment, thedose regimens range from 200-400 mg/m². In another embodiment, the doseregimens range from 250-500 mg/m². In yet another embodiment, the doseregimens range from 600-1 000 mg/m². In some embodiments the agent isadministered daily, once per week, once every other week, or once permonth. In one embodiment, a dose regimen ranging from 200-400 mg/m² isadministered once a week. In another embodiment, a dose regimen rangingfrom 250-500 mg/m² is administered once every other week.

The doses may be constant over the entire treatment period, or they mayincrease or decrease during the course of the treatment. In oneembodiment, the agent is administered once a week and starts with theadministration of 200 mg/m², and increases to 300 mg/m² and 400 mg/m² inthe second and third weeks, respectively. In another embodiment, theagent is administered once every other week and is kept constant for theentire duration of treatment with the administration of 250 mg/m². Thedoses of the agent may be administered for at least one week, at leasttwo weeks, at least three weeks, at least four weeks, at least 6 weeks,or even at least 8 weeks. Adjusting the dose of the agent within theseranges for a particular subject is well within the skill of the ordinaryclinician.

The agent may be administered via any conventional route normally usedto administer a medicament including, but not limited to, oral,parenteral (including subcutaneous, intradermal, intramuscular,intravenous, intraarticular, and intramedullary), intraperitoneal,transmucosal (including nasal), transdermal, rectal and topical(including dermal, buccal, sublingual and intraocular) routes.Intravenous delivery may take place via a bolus injection or viainfusion; infusion may be done over a period ranging from less than aminute to several hours to continuously. In certain embodiments, acourse of treatment will involve administration by a combination ofroutes.

For example, the agent may be administered via a combination ofintravenous and oral routes for the treatment of pain or anotherdisorder. In one embodiment, a “loading” dose may be administered IV inorder to bring the concentration of drug to the desired therapeuticlevel, followed by one or more maintenance doses via the oral route tokeep it there. In a further embodiment, a combination of oral and IVdelivery may be used to mitigate pain in a surgery patient. The agentmay be delivered pre-, peri-, and post-surgically by a combination of IVand oral routes. In one embodiment, the patient may be administered ormay self-administer the drug orally prior to surgery, be administeredthe drug via IV infusion during surgery and just after, and maythereafter be administered or may self-administer the drug orally aftersurgery. In another embodiment, the patient may be administered the drugIV prior to surgery, be administered the drug via IV infusion duringsurgery and just after, and may thereafter be administered or mayself-administer the drug orally after surgery.

The agent may be administered as a pharmaceutical composition in avariety of forms including, but not limited to, liquid, powder,suspensions, tablets, pills, capsules, sprays and aerosols. Thepharmaceutical compositions may include various pharmaceuticallyacceptable additives including, but not limited to, carriers,excipients, binders, stabilizers, antimicrobial agents, antioxidants,diluents and/or supports. Examples of suitable excipients and carriersare described, for example, in “Remington's Pharmaceutical Sciences,”Mack Pub. Co., New Jersey (1991). In some embodiments, the agent may beadministered via an IV infusion in an aqueous sugar solution. The agentmay also be associated with another substance that facilitates agentdelivery. For example, the agent may be associated into liposomes. Theliposomes, in turn, may be conjugated with targeting substance(s), suchas IgGFc receptors.

Formulations of the compounds disclosed herein suitable for oraladministration may be presented as discrete units such as capsules,cachets or tablets each containing a predetermined amount of the activeingredient; as a powder or granules; as a solution or a suspension in anaqueous liquid or a non-aqueous liquid; or as an oil-in-water liquidemulsion or a water-in-oil liquid emulsion. The active ingredient mayalso be presented as a bolus, electuary or paste.

Pharmaceutical preparations which can be used orally include tablets,push-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a plasticizer, such as glycerol or sorbitol. Tablets maybe made by compression or molding, optionally with one or more accessoryingredients. Compressed tablets may be prepared by compressing in asuitable machine the active ingredient in a free-flowing form such as apowder or granules, optionally mixed with binders, inert diluents, orlubricating, surface active or dispersing agents. Molded tablets may bemade by molding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent. The tablets may optionally becoated or scored and may be formulated so as to provide slow orcontrolled release of the active ingredient therein. All formulationsfor oral administration should be in dosages suitable for suchadministration. The push-fit capsules can contain the active ingredientsin admixture with filler such as lactose, binders such as starches,and/or lubricants such as talc or magnesium stearate and, optionally,stabilizers. In soft capsules, the active compounds may be dissolved orsuspended in suitable liquids, such as fatty oils, liquid paraffin, orliquid polyethylene glycols. In addition, stabilizers may be added.Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

The compounds may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampoules orin multi-dose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. The formulations may be presentedin unit-dose or multi-dose containers, for example sealed ampoules andvials, and may be stored in powder form or in a freeze-dried(lyophilized) condition requiring only the addition of the sterileliquid carrier, for example, saline or sterile pyrogen-free water,immediately prior to use. Extemporaneous injection solutions andsuspensions may be prepared from sterile powders, granules and tabletsof the kind previously described.

Formulations for parenteral administration include aqueous andnon-aqueous (oily) sterile injection solutions of the active compoundswhich may contain antioxidants, buffers, bacteriostats and solutes whichrender the formulation isotonic with the blood of the intendedrecipient; and aqueous and non-aqueous sterile suspensions which mayinclude suspending agents and thickening agents. Suitable lipophilicsolvents or vehicles include fatty oils such as sesame oil, or syntheticfatty acid esters, such as ethyl oleate or triglycerides, or liposomes.Aqueous injection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.

In addition to the formulations described previously, the compounds mayalso be formulated as a depot preparation. Such long acting formulationsmay be administered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds may be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

For buccal or sublingual administration, the compositions may take theform of tablets, lozenges, pastilles, or gels formulated in conventionalmanner. Such compositions may comprise the active ingredient in aflavored basis such as sucrose and acacia or tragacanth.

The compounds may also be formulated in rectal compositions such assuppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter, polyethylene glycol, or otherglycerides.

Certain compounds disclosed herein may be administered topically, thatis by non-systemic administration. This includes the application of acompound disclosed herein externally to the epidermis or the buccalcavity and the instillation of such a compound into the ear, eye andnose, such that the compound does not significantly enter the bloodstream. In contrast, systemic administration refers to oral,intravenous, intraperitoneal and intramuscular administration.

Formulations suitable for topical administration include liquid orsemi-liquid preparations suitable for penetration through the skin tothe site of inflammation such as gels, liniments, lotions, creams,ointments or pastes, and drops suitable for administration to the eye,ear or nose. The active ingredient for topical administration maycomprise, for example, from 0.001% to 10% w/w (by weight) of theformulation. In certain embodiments, the active ingredient may compriseas much as 10% w/w. In other embodiments, it may comprise less than 5%w/w. In certain embodiments, the active ingredient may comprise from 2%w/w to 5% w/w. In other embodiments, it may comprise from 0.1% to 1% w/wof the formulation.

For administration by inhalation, compounds may be convenientlydelivered from an insufflator, nebulizer pressurized packs or otherconvenient means of delivering an aerosol spray. Pressurized packs maycomprise a suitable propellant such as dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol, the dosageunit may be determined by providing a valve to deliver a metered amount.Alternatively, for administration by inhalation or insufflation, thecompounds according to the invention may take the form of a dry powdercomposition, for example a powder mix of the compound and a suitablepowder base such as lactose or starch. The powder composition may bepresented in unit dosage form, in for example, capsules, cartridges,gelatin or blister packs from which the powder may be administered withthe aid of an inhalator or insufflator.

Exemplary unit dosage formulations are those containing an effectivedose, as herein below recited, or an appropriate fraction thereof, ofthe active ingredient.

Fillers to be used in the compositions herein include all those nowknown and in use, as well as those developed in the future. Examples offillers, or diluents, include, without limitation, lactose, mannitol,xylitol, dextrose, sucrose, sorbitol, compressible sugar,microcrystalline cellulose (MCC), powdered cellulose, cornstarch,pregelatinized starch, dextrates, dextran, dextrin, dextrose,maltodextrin, calcium carbonate, dibasic calcium phosphate, tribasiccalcium phosphate, calcium sulfate, magnesium carbonate, magnesiumoxide, poloxamers such as polyethylene oxide, and hydroxypropyl methylcellulose. Fillers may have complexed solvent molecules, such as in thecase where the lactose used is lactose monohydrate. Fillers may also beproprietary, such in the case of the filler PROSOLV® (available from JRSPharma). PROSOLV is a proprietary, optionally high-density, silicifiedmicrocrystalline cellulose composed of 98% microcrystalline celluloseand 2% colloidal silicon dioxide. Silicification of the microcrystallinecellulose is achieved by a patented process, resulting in an intimateassociation between the colloidal silicon dioxide and microcrystallinecellulose. ProSolv comes in different grades based on particle size, andis a white or almost white, fine or granular powder, practicallyinsoluble in water, acetone, ethanol, toluene and dilute acids and in a50 g/l solution of sodium hydroxide.

Disintegrants to be used in the compositions herein include all thosenow known and in use, as well as those developed in the future. Examplesof disintegrants include, without limitation, sodium starch glycolate,sodium carboxymethyl cellulose, calcium carboxymethyl cellulose,croscarmellose sodium, povidone, crospovidone(polyvinylpolypyrrolidone), methyl cellulose, microcrystallinecellulose, powdered cellulose, low-substituted hydroxy propyl cellulose,starch, pregelatinized starch, and sodium alginate.

Lubricants to be used in the compositions herein include all those nowknown and in use, as well as those developed in the future. Examples oflubricants include, without limitation, calcium stearate, glycerylmonostearate, glyceryl palmitostearate, hydrogenated vegetable oil,light mineral oil, magnesium stearate, mineral oil, polyethylene glycol,sodium benzoate, sodium lauryl sulfate, sodium stearyl fumarate, stearicacid, talc, and zinc stearate.

Glidants to be used in the compositions herein include all those nowknown and in use, as well as those developed in the future. Examples ofglidants include, without limitation, silicon dioxide (SiO₂), talccornstarch, and poloxamers. Poloxamers (or LUTROL®, available from theBASF Corporation) are A-B-A block copolymers in which the A segment is ahydrophilic polyethylene glycol homopolymer and the B segment ishydrophobic polypropylene glycol homopolymer.

Tablet binders to be used in the compositions herein include all thosenow known and in use, as well as those developed in the future. Examplesof tablet binders include, without limitation, acacia, alginic acid,carbomer, carboxymethyl cellulose sodium, dextrin, ethylcellulose,gelatin, guar gum, hydrogenated vegetable oil, hydroxyethylcellulose,hydroxypropyl cellulose, hydroxypropylmethyl cellulose, copolyvidone,methyl cellulose, liquid glucose, maltodextrin, polymethacrylates,povidone, pregelatinized starch, sodium alginate, starch, sucrose,tragacanth, and zein.

Examples of surfactants include, without limitation, fatty acid andalkyl sulfonates; commercial surfactants such as benzethanium chloride(HYAMINE® 1622, available from Lonza, Inc., Fairlawn, N.J.); DOCUSATESODIUM® (available from Mallinckrodt Spec. Chem., St. Louis, Mo.);polyoxyethylene sorbitan fatty acid esters (TWEEN®, available from ICIAmericas Inc., Wilmington, Del.; LIPOSORB® P-20, available from LipochemInc., Patterson N.J.; CAPMUL® POE-0, available from Abitec Corp.,Janesville, Wis.), polyoxyethylene (20) sorbitan monooleate (TWEEN 80®,available from ICI Americas Inc., Wilmington, Del.); and naturalsurfactants such as sodium taurocholic acid,1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, lecithin, and otherphospholipids and mono- and diglycerides. Such materials canadvantageously be employed to increase the rate of dissolution byfacilitating wetting, thereby increasing the maximum dissolvedconcentration, and also to inhibit crystallization or precipitation ofdrug by interacting with the dissolved drug by mechanisms such ascomplexation, formation of inclusion complexes, formation of micelles oradsorbing to the surface of solid drug

Drug complexing agents and solubilizers to be used in the compositionsherein include all those now known and in use, as well as thosedeveloped in the future. Examples of drug complexing agents orsolubilizers include, without limitation, the polyethylene glycols,caffeine, xanthene, gentisic acid and cylodextrins.

The addition of pH modifiers such as acids, bases, or buffers may alsobe beneficial, retarding or enhancing the rate of dissolution of thecomposition, or, alternatively, helping to improve the chemicalstability of the composition. Suitable pH modifiers to be used in thecompositions herein include all those now known and in use, as well asthose developed in the future.

It should be understood that in addition to the ingredients particularlymentioned above, the formulations provided herein may include otheragents conventional in the art having regard to the type of formulationin question. Proper formulation is dependent upon the route ofadministration chosen. Any of the well-known techniques, carriers, andexcipients may be used as suitable and as understood in the art; e.g.,Remington, supra. The pharmaceutical compositions may be manufactured ina manner that is itself known, e.g., by means of conventional mixing,dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping or compression processes.

Compounds may be administered orally or via injection at a dose of from0.1 to 500 mg/kg per day. The dose range for adult humans is generallyfrom 5 mg to 2 g/day. Tablets or other forms of presentation provided indiscrete units may conveniently contain an amount of one or morecompounds which is effective at such dosage or as a multiple of thesame, for instance, units containing 5 mg to 500 mg, usually around 10mg to 200 mg.

The precise amount of compound administered to a subject will be theresponsibility of the attendant physician. The specific dose level forany particular subject will depend upon a variety of factors includingthe activity of the specific compound employed, the age, body weight,general health, sex, diets, time of administration, route ofadministration, rate of excretion, drug combination, the precisedisorder being treated, and the severity of the indication or conditionbeing treated. Also, the route of administration may vary depending onthe condition and its severity. Dosing frequency may also be selected oradjusted based on factors including those above as well as theformulation of the compound delivered. Dosing may occur, for example:once daily, twice daily, three or four times daily, every other day,weekly, bi-weekly, or monthly; or in cycles comprising a sustaineddosing period followed by a non-dosing period; or on an as-needed basis.

In certain instances, it may be appropriate to administer at least oneof the compounds described herein (or a pharmaceutically acceptablesalt, ester, or prodrug thereof) in combination with another therapeuticagent. By way of example only, if one of the side effects experienced bya subject upon receiving one of the compounds herein is hypertension,then it may be appropriate to administer an anti-hypertensive agent incombination with the initial therapeutic agent. Or, by way of exampleonly, the therapeutic effectiveness of one of the compounds describedherein may be enhanced by administration of an adjuvant (i.e., by itselfthe adjuvant may only have minimal therapeutic benefit, but incombination with another therapeutic agent, the overall therapeuticbenefit to the subject is enhanced). Or, by way of example only, thebenefit experienced by a subject may be increased by administering oneof the compounds described herein with another therapeutic agent (whichalso includes a therapeutic regimen) that also has therapeutic benefit.By way of example only, in a treatment for neuropathy involvingadministration of one of the compounds described herein, increasedtherapeutic benefit may result by also providing the subject withanother therapeutic agent for neuropathy. In any case, regardless of thedisease, disorder or condition being treated, the overall benefitexperienced by the subject may simply be additive of the two therapeuticagents or the subject may experience a synergistic benefit.

In certain embodiments, the other therapeutic agent is an antiviralagent. In one embodiment, the antiviral agent is an antiretroviralagent, e.g. nucleoside reverse transcriptase inhibitors, nucleotidereverse transcriptase inhibitors, nonnucleoside reverse transcriptaseinhibitors, protease inhibitors, entry inhibitors, integrase inhibitorsor gp41, CXCR4, or gp120 inhibitors. Examples of nucleoside reversetranscriptase inhibitors for the treatment of HIV infections includeamdoxovir, elvucitabine, alovudine, racivir (±-FTC), phosphazide,fozivudine tidoxil, apricitibine (AVX754), amdoxovir, zidovudine (AZT),didanosine (ddl), lamivudine (3TC), stavudine (d4T), zalcitabine (ddC),emtricitabine (FTC), and abacavir (ABC). Examples of nucleotide reversetranscriptase inhibitors include tenofovir (TDF) and adefovir. Examplesof non-nucleoside reverse transcriptase inhibitors include capravirine,emivirine, calanolide A, etravirine, efavirenz (EFV), nevirapine (NVP)and delavirdine (DLV). Examples of protease inhibitors includeamprenavir (APV), tipranavir (TPV), lopinavir (LPV), fosamprenavir(FPV), atazanavir (ATV), darunavir, brecanavir, mozenavir, indinavir(IDV), nelfinavir (NFV), ritonavir (RTV), and saquinavir (SQV). Examplesof entry inhibitors include SPOIA. Examples of a HIV integrase inhibitorinclude curcumin, derivatives of curcumin, chicoric acid, derivatives ofchicoric acid, 3,5-dicaffeoylquinic acid, derivatives of3,5dicaffeoylquinic acid, aurintricarboxylic acid, derivatives ofaurintricarboxylic acid, caffeic acid phenethyl ester, derivatives ofcaffeic acid phenethyl ester, tyrphostin, derivatives of tyrphostin,quercetin, derivatives of quercetin, S-1360, zintevir (AR-177),L-870812, and L-25 870810, MK-0518, BMS-538158, GSK364735C, Examples ofa gp41 inhibitor include enfuvirtide (ENF). Examples of a CXCR4inhibitor include AMD-070, Examples of a gp120 inhibitor includeBMS-488043.

In another embodiment, the polyamine analog is administered concurrentlywith a highly active antiretroviral therapy (HAART), i.e., a combinationof a protease inhibitor, a non-nucleoside reverse transcriptaseinhibitor and a nucleoside reverse transcriptase inhibitor, or acombination of two non-nucleoside reverse transcriptase inhibitors and anucleoside reverse transcriptase inhibitor. In general, the polyamineanalog may be administered simultaneously or sequentially (i.e., beforeor after) with the administration of antiviral or antiretroviral agents.Administration of the antiviral and antiretroviral agents to subjects inneed thereof can be made in accordance with regimens and dosages wellknown in the art.

In yet other embodiments, the antiviral agent is an agent that iscapable of reducing the immunodeficiency viral load in T-cells. T-cells,particularly CD4+ T-cells, also serve as a viral reservoir forimmunodeficiency viruses such as HIV. Thus, combination treatments ofpolyamine analogs with agents that reduce the immunodeficiency viralload in T-cells are particularly desirable for flushing or destroyingviral reservoirs of immunodeficiency virus. Suitable agents that reducethe immunodeficiency viral load in T-cells are reviewed in Pierson etal. (Annu. Rev. Immunol. (2000), 18:665-708) and include, withoutlimitation, T-cell activating cytokines, anti-CD3 antibodies, andanti-CD45RO-toxin conjugates. For example, T-cell activating cytokinesuch as IL-2, IL-6, TNF-α, and any two or more combinations thereof maybe used in the present methods.

In other embodiments, the other therapeutic agent is a TNF inhibitor.The TNF inhibitor may be: a monoclonal antibody such as, for example,infliximab (Remicade), adalimumab (Humira), certolizumab pegol (Cimzia),or golimumab (Simponi); a circulating receptor fusion protein such asetanercept (Enbrel); or a small molecule, such as pentoxifylline orbupropion (Zyban, Wellbutrin).

In other embodiments, the other therapeutic agent is a disease-modifyinganti-rheumatic drug (DMARD). Examples of DMARDs include azathioprine,ciclosporin (cyclosporine A), D-penicillamine, gold salts,hydroxychloroquine, leflunomide, methotrexate (MTX), minocycline,sulfasalazine (SSZ), and cyclophosphamide.

In further embodiments, the other therapeutic agent is methotrexate.

Other agents for used in combination include interleukin 1 (IL-1)blockers such as anakinra (Kineret), T-cell costimulation blockers suchas abatacept (Orencia), interleukin 6 (IL-6) blockers such astocilizumab (an anti-IL-6 receptor antibody; RoActemra, Actemra),monoclonal antibodies against B cells such as rituximab (Rituxan), andother biologics (eg. Ocrelizumab, Ofatumumab, Golimumab, andCertolizumab pegol).

In other embodiments, the other therapeutic agent is a glucocorticoid ora non-steroidal anti-inflammatory drug (NSAID). NSAIDS include propionicacid derivatives such as ibuprofen, naproxen, fenoprofen, ketoprofen,flurbiprofen, and oxaprozin; acetic acid derivatives such asindomethacin, sulindac, etodolac, and diclofenac; enolic acid (oxicam)derivatives such as piroxicam and meloxicam; fenamic acid derivativessuch as mefenamic acid and meclofenamic acid; selective COX-2 inhibitors(Coxibs) such as celecoxib (Celebrex), rofecoxib, valdecoxib, parecoxib,lumiracoxib, and etoricoxib.

In any case, the multiple therapeutic agents (at least one of which is acompound disclosed herein) may be administered in any order or evensimultaneously. If simultaneously, the multiple therapeutic agents maybe provided in a single, unified form, or in multiple forms (by way ofexample only, either as a single pill or as two separate pills). One ofthe therapeutic agents may be given in multiple doses, or both may begiven as multiple doses. If not simultaneous, the timing between thedoses of the multiple therapeutic agents may be any duration of timeranging from a few minutes to four weeks.

Thus, in another aspect, certain embodiments provide methods fortreating disorders in a human or animal subject in need of suchtreatment comprising administering to said subject an amount of acompound disclosed herein effective to reduce or prevent said disorderin the subject, optionally in combination with at least one additionalagent for the treatment of said disorder that is known in the art.Specific diseases to be treated by the compounds, compositions, andmethods disclosed herein, singly or in combination, include, withoutlimitation: pain; neuropathy; inflammation and related disorders;arthritis; metabolic inflammatory disorders; respiratory disorders;autoimmune disorders; neurological disorders; and proliferativedisorders, including cancer and non-cancerous diseases.

The compounds disclosed herein are useful to treat patients with pain,including neuropathy and/or neuropathic pain, and inflammatory pain.Pain indications include, but are not limited to, treatment orprophylaxis of surgical or post-surgical pain for various surgicalprocedures including amputation, post-cardiac surgery, dentalpain/dental extraction, pain resulting from cancer, muscular pain,mastalgia, pain resulting from dermal injuries, lower back pain,headaches of various etiologies, including migraine, menstrual cramps,and the like. The compounds are also useful for the treatment ofpain-related disorders such as tactile allodynia and hyperalgesia. Thepain may be somatogenic (either nociceptive or neuropathic), acuteand/or chronic. Peripheral neuropathies which can be treated with thecompounds disclosed herein include mono-neuropathies, mono-multiplexneuropathies, and poly-neuropathies, including axonal and demyelinatingneuropathies. Both sensory and motor neuropathies are encompassed. Theneuropathy or neuropathic pain may be associated with a number ofperipheral neuropathies of varying etiologies, including but not limitedto:

-   -   trauma-induced neuropathies, including those caused by physical        injury (such as blunt trauma, abrasion, or burns) or disease        state, physical damage to the brain, physical damage to the        spinal cord, or stroke associated with brain damage;        neurological disorders related to neurodegeneration; and        post-surgical neuropathies and neuropathic pain (such as from        tumor resection, mastectomy, and the like)    -   infectious and viral neuropathies, including those caused by        leprosy, Lyme disease, a herpes virus (and more particularly by        a herpes zoster virus, which may lead to post-herpetic        neuralgia), human immunodeficiency virus (HIV, which may lead to        HIV neuropathy), or a papilloma virus, or any other        pathogen-induced nerve damage;    -   toxin-induced neuropathies (including but not limited to        neuropathies induced by alcoholism, vitamin B6 intoxication,        hexacarbon intoxication, amiodarone, chloramphenicol,        disulfiram, isoniazide, gold, lithium, metronidazole,        misonidazole, nitrofurantoin);    -   drug-induced neuropathies, including therapeutic-drug-induced        neuropathy, particularly a) chemotherapy-induced neuropathies        caused by anti-cancer agents such as taxol, taxotere, cisplatin,        nocodazole, vincristine, vindesine and vinblastine, and b)        anti-viral neuropathies caused by anti-viral agents such as ddI,        DDC, d4T, foscarnet, dapsone, metronidazole, and isoniazid);    -   vitamin-deficiency-induced neuropathies including those        resulting from vitamin B12 deficiency, vitamin B6 deficiency,        and vitamin E deficiency);    -   hereditary neuropathy (including but not limited to Friedreich        ataxia, familial amyloid polyneuropathy, Tangier disease, Fabry        disease;    -   diabetic neuropathy and neuropathy caused by metabolic disorders        such as renal insufficiency and hypothyroidism;    -   neuropathy secondary to tumor infiltration,    -   auto-immune neuropathies, including those resulting from        Guillain-Barre syndrome, chronic inflammatory de-myelinating        polyneuropathy, monoclonal gammopathy of undetermined        significance and polyneuropathy, and multiple sclerosis;    -   other neuropathies and neuropathic pain syndromes including        inflammation-induced nerve damage, neurodegeneration,        post-traumatic neuralgia, central neuropathic pain syndromes        such as phantom limb pain, pain, complex regional pain syndromes        (including but not limited to reflex sympathetic dystrophy,        causalgia), neoplasia-associated pain, vasculitic/angiopathic        neuropathy, and sciatica; and    -   idiopathic neuropathies,

In certain embodiments, neuropathic pain may alternatively be manifestedas allodynia, hyperalgesic pain, thermal hyperalgesia, or phantom pain.In another embodiment, neuropathy may instead lead to loss of painsensitivity. Additional sub-categories of neuropathic pain are discussedin Dworkin, Clin J Pain (2002) vol. 18(6) pp. 343-9.

Furthermore, the compounds disclosed herein can be used in the treatmentor prevention of opiate tolerance in patients needing protracted opiateanalgesics, and benzodiazepine tolerance in patients takingbenzodiazepines, and other addictive behavior, for example, nicotineaddiction, alcoholism, and eating disorders. Moreover, the compoundsdisclosed herein are useful in the treatment or prevention of drugwithdrawal symptoms, for example treatment or prevention of symptoms ofwithdrawal from opiate, alcohol, or tobacco addiction.

The compounds disclosed herein are useful in therapeutic methods totreat or prevent respiratory disease or conditions, includingtherapeutic methods of use in medicine for preventing and treating arespiratory disease or condition including: asthmatic conditionsincluding allergen-induced asthma, exercise-induced asthma,pollution-induced asthma, cold-induced asthma, and viral-induced-asthma;chronic obstructive pulmonary diseases including chronic bronchitis withnormal airflow, chronic bronchitis with airway obstruction (chronicobstructive bronchitis), emphysema, asthmatic bronchitis, and bullousdisease; and other pulmonary diseases involving inflammation includingbronchioectasis cystic fibrosis, hypersensitivity pneumonitis, farmer'slung, acute respiratory distress syndrome, pneumonia, aspiration orinhalation injury, fat embolism in the lung, acidosis inflammation ofthe lung, acute pulmonary edema, acute mountain sickness, acutepulmonary hypertension, persistent pulmonary hypertension of thenewborn, perinatal aspiration syndrome, hyaline membrane disease, acutepulmonary thromboembolism, heparin-protamine reactions, sepsis, statusasthamticus, hypoxia, hyperoxic lung injuries, and injury induced byinhalation of certain injurious agents including cigarette smoking,leading up to complications thereof such as lung carcinoma.

Other disorders or conditions which can be advantageously treated by thecompounds disclosed herein include inflammation and inflammatoryconditions. Inflammatory conditions include, without limitation:arthritis, including sub-types and related conditions such as rheumatoidarthritis, spondyloarthropathies, gouty arthritis, osteoarthritis,systemic lupus erythematosus, juvenile arthritis, acute rheumaticarthritis, enteropathic arthritis, neuropathic arthritis, psoriaticarthritis, and pyogenic arthritis; osteoporosis, tendonitis, bursitis,and other related bone and joint disorders; gastrointestinal conditionssuch as reflux esophagitis, diarrhea, inflammatory bowel disease,Crohn's disease, gastritis, irritable bowel syndrome, ulcerativecolitis, acute and chronic inflammation of the pancreas; pulmonaryinflammation, such as that associated with viral infections and cysticfibrosis; skin-related conditions such as psoriasis, eczema, burns,sunburn, dermatitis (such as contact dermatitis, atopic dermatitis, andallergic dermatitis), and hives; pancreatitis, hepatitis, pruritis andvitiligo. In addition, compounds of invention are also useful in organtransplant patients either alone or in combination with conventionalimmunomodulators.

Autoimmune disorders may be ameliorated by the treatment with compoundsdisclosed herein. Autoimmune disorders include Crohns disease,ulcerative colitis, dermatitis, dermatomyositis, diabetes mellitus type1, Goodpasture's syndrome, Graves' disease, Guillain-Barré syndrome(GBS), autoimmune encephalomyelitis, Hashimoto's disease, idiopathicthrombocytopenic purpura, lupus erythematosus, mixed connective tissuedisease, multiple sclerosis (MS), myasthenia gravis, narcolepsy,pemphigus vulgaris, pernicious anemia, psoriasis, psoriatic arthritis,polymyositis, primary biliary cirrhosis, rheumatoid arthritis, Sjögren'ssyndrome, scleroderma, temporal arteritis (also known as “giant cellarteritis”), vasculitis, and Wegener's granulomatosis. The compoundsdisclosed herein may regulate TH-17 (T-helper cells producinginterleukin 17) cells or IL-17 levels.

In addition, the compounds disclosed herein can be used to treatmetabolic disorders that are typically associated with an exaggeratedinflammatory signaling, such as insulin resistance, diabetes (type I ortype II), metabolic syndrome, nonalcoholic steatohepatitis,atherosclerosis, cardiovascular disease, congestive heart failure,myocarditis, atherosclerosis, and aortic aneurysm.

The compounds disclosed herein are also useful in treating organ andtissue injury associated with severe burns, sepsis, trauma, wounds, andhemorrhage- or resuscitation-induced hypotension, and also in suchdiseases as vascular diseases, migraine headaches, periarteritis nodosa,thyroiditis, aplastic anemia, Hodgkin's disease, sclerodoma, rheumaticfever, type I diabetes, neuromuscular junction disease includingmyasthenia gravis, white matter disease including multiple sclerosis,sarcoidosis, nephritis, nephrotic syndrome, Behcet's syndrome,polymyositis, gingivitis, periodontis, swelling occurring after injury,ischemias including myocardial ischemia, cardiovascular ischemia, andischemia secondary to cardiac arrest, and the like.

The compounds of the subject invention are also useful for the treatmentof certain diseases and disorders of the nervous system. Central nervoussystem disorders in which nitric oxide inhibition is useful includecortical dementias including Alzheimer's disease, central nervous systemdamage resulting from stroke, ischemias including cerebral ischemia(both focal ischemia, thrombotic stroke and global ischemia (forexample, secondary to cardiac arrest), and trauma. Neurodegenerativedisorders in which nitric oxide inhibition is useful include nervedegeneration or nerve necrosis in disorders such as hypoxia,hypoglycemia, epilepsy, and in cases of central nervous system (CNS)trauma (such as spinal cord and head injury), hyperbaric oxygen-inducedconvulsions and toxicity, dementia e.g. pre-senile dementia, andAIDS-related dementia, cachexia, Sydenham's chorea, Huntington'sdisease, Parkinson's Disease, amyotrophic lateral sclerosis (ALS),Korsakoffs disease, cognitive disorders relating to a cerebral vesseldisorder, hypersensitivity, sleeping disorders, schizophrenia,depression, depression or other symptoms associated with PremenstrualSyndrome (PMS), and anxiety.

Still other disorders or conditions advantageously treated by thecompounds of the subject invention include the prevention or treatmentof (hyper) proliferative diseases, especially cancers, either alone orin combination of standards of care especially those agents that targettumor growth by re-instating the aberrant apoptotic machinery in themalignant cells. Hematological and non-hematological malignancies whichmay be treated or prevented include but are not limited to multiplemyeloma, acute and chronic leukemias including Acute LymphocyticLeukemia (ALL), Chronic Lymphocytic Leukemia (CLL), and ChronicMyelogenous Leukemia (CLL), lymphomas, including Hodgkin's lymphoma andnon-Hodgkin's lymphoma (low, intermediate, and high grade), as well assolid tumors and malignancies of the brain, head and neck, breast, lung,reproductive tract, upper digestive tract, pancreas, liver, renal,bladder, prostate and colorectal. The present compounds and methods canalso be used to treat the fibrosis, such as that which occurs withradiation therapy. The present compounds and methods can be used totreat subjects having adenomatous polyps, including those with familialadenomatous polyposis (FAP). Additionally, the present compounds andmethods can be used to prevent polyps from forming in patients at riskof FAP. Non-cancerous proliferative disorders additionally includepsoriasis, eczema, and dermatitis.

Compounds disclosed herein may also be used in the treatment ofpolycystic kidney disease, as well as other diseases of renaldysfunction.

The compounds of the subject invention can be used in the treatment ofophthalmic diseases, such as glaucoma, retinal ganglion degeneration,ocular ischemia, corneal neovascularization, optic neuritis, retinitis,retinopathies such as glaucomatous retinopathy and/or diabeticretinopathy, uveitis, ocular photophobia, dry eye, Sjogren's syndrome,seasonal and chronic allergic conjunctivitis, and of inflammation andpain associated with chronic ocular disorders and acute injury to theeye tissue. The compounds can also be used to treat post-operativeinflammation or pain as from ophthalmic surgery such as cataract surgeryand refractive surgery.

The present compounds may also be used in co-therapies, partially orcompletely, in place of other conventional anti-inflammatory therapies,such as together with steroids, NSAIDs, COX-2 selective inhibitors,5-lipoxygenase inhibitors, LTB₄ antagonists and LTA₄ hydrolaseinhibitors. The compounds of the subject invention may also be used toprevent tissue damage when therapeutically combined with antibacterialor antiviral agents.

It has been shown that osteopontin (“OPN”) can be regulated byregulating AMD-I, polyamine biosynthesis, adenosine or a pathwaycontaining either AMD-I or adenosine. Osteopontin, also known assecreted phosphoprotein 1 (“SPP1”), early T-lymphocyte activation marker(“Eta-I”), sialoprotein I or 44K BPP (bone phosphoprotein), is aglycosylated phosphoprotein found in plasma, other bodily fluids, andextracellular matrices. The protein is composed of approximately 300amino acids residues and has about 30 carbohydrate residues, including10 sialic acid residues, attached to it. OPN is an acidic protein whichexhibits a high amino acid homology between species (e.g., mouse, rat,human and pig) with several conserved elements including a stretch of 7to 9 Asp or Glu residues.

Osteopontin is biosynthesized by a variety of tissue types includingpreosteoblasts, osteoblasts, osteocytes, extraosseous cells in the innerear, brain, kidney, deciduum, placenta, odontoblasts, some bone marrowcells, hypertrophic chondrocytes, macrophages, smooth muscle, andendothelial cells. In the bone, the protein is primarily made by cellsof the osteoblastic lineage and deposited on mineralized matrix. It isabundant in bone mineral matrix and accelerates bone regeneration andremodeling. Osteopontin is a multifunctional protein with an ability tobind several proteins, including integrin proteins and variants of theprotein CD44.

Osteopontin is associated with, and plays a role in, the regulation andprogression of many diseases. OPN is known to be increased in a numberof autoimmune disorders and is overexpressed in a variety of cancers.Plasma levels of OPN are also elevated in individuals with coronaryartery disease and elevated levels of OPN are found in the synovialfluid of individuals with rheumatoid arthritis. Modulation of OPN may,therefore, confer significant therapeutic benefits to subjects havingthese conditions. Accordingly, disclosed herein is a method formodulating OPN levels in a subject, comprising administering to thesubject an amount of a compound of the invention, e.g., MGBG, sufficientto modulate OPN. Such modulation may be of total body or plasma OPN, orit may be of OPN in a target organ or tissue. In certain embodiments,said modulation is downwards, i.e., leading to reduced levels of OPN orreduced activity of OPN.

Additionally, MGBG has been examined in HIV and related chronic andprogressive conditions such as HIV neuropathy and HIV dementia. In thecase of HIV and related disorders and complications, polyamine analogsor polyamine biosynthesis inhibitors such as MGBG may work via anadditional mechanism. HIV is an RNA retrovirus, that upon successfulinfection of a host cell, reverse transcribes its genomic RNA into DNA,which then, in a double stranded form, integrates into susceptible hostcells. The major targets for infection in vivo are the CD4 expressing Tcells and macrophages. Whereas T cells, upon activation of the HIV DNAinto an infectious RNA form, generally get killed, the virus-expressingmacrophages persist after infection and likely serve as the long termHIV DNA reservoir in vivo.

Several studies have confirmed the long lived nature of HIV infectedmacrophages in vivo, and the consequences for subjects with AIDS-relatedneurological conditions. At least one study on the HIV reservoir hasprovided half life estimates of 4 years for infected blood macrophagesand less than 2 years for infected T cells. Both values help explain thereason for the failure of highly active antiretroviral therapy (HAART)to clear the virus in vivo. Additionally, studies on the HIV DNAsequence in vivo showed that in HIV plasma viral load negative subjectson HAART HIV replication continued to occur in vivo within macrophagesbut not T cells. Therefore, the longest lived reservoir of HIV in vivois the macrophage. Furthermore, one study showed that the ancestral formof HIV in vivo in a subject who died of AIDS related dementia residedwithin macrophages in the outer membrane covering of the brain(meningeal layer). Viral sequences present in this long lived reservoirgave rise to all of the sequences residing in other portions of thebrain as well as the peripherally located seminal vesicles and lymphnodes. Yet another study has suggested a mechanism for the long livednature of HIV infected macrophages. This study mapped HIV DNA insertionsites within macrophages in tissues from subjects with late stage AIDS.All of the insertion sites were within genes near activation geneticloci that, if activated through an HIV insertional process, would keepthe infected macrophages in a persistently activated and essentiallyimmortal state.

Ideally, drugs would target only HIV-infected macrophages and sparenormal macrophages. In fact, it has been shown that in AIDS dementiasubjects, infected macrophages express CD14 as well as CD16 and elevatedlevels of the activation marker, HLA-DR, as well as the proliferationmarker, proliferating cell nuclear antigen (PCNA). Polyamine analogs andpolyamine biosynthesis inhibitors, such as MGBG, can be used to decreasethe proviral load in a subject by destroying macrophages that act asproviral reservoirs.

Exemplary embodiments of the present methods are provided in thefollowing examples. The following examples are presented to illustratethe methods of the invention and to assist one of ordinary skill inusing the same, and are not to be construed as limiting the scope of theinvention.

MGBG Oral Activity Assays

The following standard abbreviations are used to represent theassociated pharmacokinetic parameters.

-   -   AUC Area under the curve up to the last measurable concentration        plus the AUC extrapolated from the last measurable concentration        (C_(last) at t_(last)) to infinity:        AUC_(INFobs)=AUC_(0-tlast)+C_(last)/Lambda z (where λz is the        first order rate constant associated with the terminal        (log-linear) portion of the curve)    -   AUC₀₋₁₂ Area under the curve between the time of dose and the 12        h time point    -   AUC₀₋₂₄ Area under the curve between the time of dose and the 24        h time point    -   F Fraction available (bioavailability):        F=[AUC_(oral)]·dose_(iv)/[AUC_(iv)]·dose_(oral)    -   Cl_(obs) Observed clearance    -   Vss_(obs) Steady state volume of distribution    -   V_(d) Volume of distribution (often used with oral)    -   Cl/F_(obs) Apparent total body clearance as a function of        bioavailability    -   t_(1/2) Terminal half-life (HL_(λz))    -   C_(max) The maximum observed concentration    -   T_(max) The time at which C_(max) occurred        Rhesus Macaque Single-Dose

Two groups of three male rhesus monkeys were fasted overnight beforebeing administered the test article, MGBG, as either a single bolusintravenous dose of 1 mg/kg (Group 1) or as a single oral gavage dose of10 mg/kg (Group 2). Dose formulation analysis verified administered dosesolutions as within 14% of targeted concentrations of 1 and 10 mg/kg forGroups 1 and 2, respectively.

Blood samples were collected into tubes containing lithium heparin fromthe femoral vein/artery (approximately 1.0 mL) for plasma MGBGconcentration measurement from all intravenously dosed animals prior todosing and at approximately T=0.083 (5 min), 0.25 (15 min), 0.5 (30min), 1, 2, 4, 8, and 24 hours after dosing. Blood samples for plasmaMGBG concentration measurement were collected from all orally dosedanimals prior to dosing at approximately T=1, 2, 4, 8, 12, 24, and 36hours after dosing. Food was also withheld through the first four hoursof blood sample collection.

The samples were centrifuged under refrigerated conditions followingcompletion of sample collection at each interval. The resulting plasmawas separated and stored frozen at approximately −70° C. until analysis.

PK analysis was performed on the individual plasma concentration-timeprofiles for MGBG using the WinNonlin non-compartmental approach (lineartrapezoidal rule for AUC calculations). Nominal dose values and samplingtimes were used for calculations. All MGBG plasma concentrationmeasurements reported as BQL (<2.51 ng/mL) were set equal to zero forthe purpose of analysis. Following IV and PO administration of MGBG,plasma PK disposition parameters were calculated using the WinNonlindefault selection criteria for the selection of the Lambda Z.

Evidence of systemic plasma MGBG exposure was observed at all collectedplasma time points following IV and PO administration of MGBG. Hemolysiswas noted in one animal in Group 1 at a single time point, which mayhave negatively impacted the MGBG plasma concentration analysis for thisanimal. Consequently, a model-dependent two-compartmental analysis wasused to calculate bioavailability.

Dog Single-Dose

Two groups of three male beagle dogs weighing 9.0-10.7 kg and aged 8-30months were fasted overnight before being administered the test article,MGBG, as either a single bolus intravenous dose of 1 mg/kg (Group 1) oras a single oral gavage dose of 10 mg/kg (Group 2). Dose formulationanalysis verified administered dose solutions as within 17% of targetedconcentrations of 1 and 10 mg/kg for Groups 1 and 2, respectively.

Blood samples (approximately 2.0 mL) were collected for plasma MGBGconcentration measurement from all intravenously dosed animals prior todosing and at approximately T=0.083 (5 min), 0.25 (15 min), 0.5 (30min), 1, 2, 4, 8, and 24 hours after dosing. A similar procedure wasused with orally dosed animals was used, except that collection tookplace at T=1, 2, 4, 8, 12, 24, and 36 hours after dosing. The sampleswere centrifuged under refrigerated conditions following completion ofsample collection at each interval. The resulting plasma was separatedand stored frozen at approximately −70° C. until analysis.

Analysis was performed by LC/MS/MS, and plasma PK disposition parameterswere calculated using the last five plasma concentrations for IV (1-24h) and PO (4-36 h) administration for the selection of the Lambda Z. Dueto inter-animal variability and limited terminal phase data, theseresults should be interpreted with caution.

No clinically abnormal findings followed IV or oral administration.Systemic exposure was observed at all time points.

Rat Single-Dose

Eighteen male Sprague Dawley rats (Charles River) weighing 217-263 g andaged 8-9 weeks were administered the test article, MGBG as either asingle bolus intravenous dose of 1 mg/kg (Group 1) or as a single oralgavage dose of 10 mg/kg. A cohort of three animals was sacrificed viaCO₂ inhalation anaesthesia after final blood collection at each of T=2,4, 12, 24, 36, and 48 hours post-dose. Dose formulation analysisverified administered dose solutions as within 17% of targetedconcentration of 10 mg/kg.

Analysis was performed by LC/MS/MS. Pharmacokinetic analyses wereperformed on the mean MGBG plasma concentration versus time data usingthe non-compartmental approach (linear trapezoidal rule for AUCcalculations). The WinNonlin sparse sampling tool was used for PKcalculations. All samples reported as BQL (Below the Limit ofQuantitation, in plasma 2.50 ng/mL) were changed to 0.00 ng/mL for thepurpose of analysis. Dose formulation analysis revealed thatformulations were within 15% of the targeted dose concentration of 10mg/kg.

Abnormal clinical findings were not noted following dosing. A single POadministration of 10 mg/kg of MGBG resulted in evidence of measurableMGBG levels in plasma through the 12 hour time point; beyond that point,certain samples began measure BLQ.

Mouse Single-Dose

Twenty-four male DBA/1 mice weighing 19.5-24.7 g and aged 7-9 weeksadministered the test article, MGBG, as either a single bolusintravenous dose via a lateral tail vein of 1 mg/kg (Group 1, n=12) oras a single oral gavage dose of 10 mg/kg (Group 2, n=12). Each dosegroup consisted of 4 cohorts of 3 animals each. Group 1 was sampled at5, 15, and 30 minutes after dosing; and 1, 2, 4, 8, and 24 hours afterdosing. Group 2 was sampled at 1, 2, 4, 8, 12, 24, and 36 hours afterdosing. Starting with the first time point, a new cohort was sampled ateach successive time point up to the 1-hour (Group 1) or 12-hour (Group2) time point. The order of sampling among the cohorts was repeated forthe subsequent time points (some cohorts may have been bled only once).The second bleed for each cohort was terminal. Animals were sacrificedvia CO₂ inhalation anaesthesia after final blood collection.

The samples were centrifuged under refrigerated conditions followingcompletion of sample collection at each interval. The resulting plasmawas separated and stored frozen at approximately −70° C. until analysis.Analysis was performed by LC/MS/MS. Pharmacokinetic analyses wereperformed on the mean MGBG plasma concentration versus time data usingthe non-compartmental approach (linear trapezoidal rule for AUCcalculations). The WinNonlin sparse sampling tool was used for PKcalculations. Dose formulation analysis revealed that the IV and POformulations were within 15% of their targeted concentrations.

Abnormal clinical findings were not noted following dosing. Evidence ofsystemic plasma MGBG exposure was observed at all collected plasma timepoints following IV and PO administration of MGBG.

Results of the foregoing assays are shown below in Tables 2 and 3.Values reported are mean across treatment groups without standarddeviation.

TABLE 2 Cl_(obs) AUC t_(1/2) T_(max) C_(max) (mL/ V_(SSobs) (h * ng/ IV(h) (h) (ng/mL) min/kg) (L/kg) mL) rhesus 30.8 0.139 757 13.7 13.2 1660rat 17.4 0.083 684 13.4 15.4 1250 mouse 13 0.083 181 49.6 38.3 336 dog15.8 0.083 1180 13.7 14.6 1410

TABLE 3 AUC Cl_(obs) t_(1/2) T_(max) C_(max) (h * ng/ (mL/ V_(d) ORAL(h) (h) (ng/mL) mL) min/kg) (L/kg) F % rhesus 24.2 3.33 192 4240 6.6313.2 35.0% rat 28.1 4.67 55.8  1280** 3.18 15.4 11.6% mouse 11.8 1 1061420 6.68 38.3 44.3% dog 15.5 1 616 6290 8.29 14.6 49.0%

In Table 2 above, the double asterisk indicates that the rat AUCreported is the AUC_(all), computed from time zero to the time of thelast plasma concentration measurement. Each of these values carries thecaveat that terminal measurements are subject to different methods ofextrapolation.

Multi-Dose Rat Pharmacokinetic and Tolerability Study

The purpose of this study was to determine the pharmacokinetic (PK)properties and tolerability of MGBG in rats. Additionally, recovery fromany toxic effects was assessed after a seven day non-dosing period.Tolerability was demonstrated in test article-treated animals by bodyweight changes similar to the control group and a lack of adverseclinical observations.

Three per group of male Sprague Dawley (CD® MS, Charles River) aged 7-9weeks and weighing 222.7-252.0 g were administered by oral (PO) gavage,twice daily, at 10, 20, or 30 mg/kg/dose (20, 40, or 60 mg/kg/day) forseven consecutive days. A washout period of seven days followed.Collection of approximately 200 μL of whole blood was collected from thetail vein of all animals in Groups 5, 6, and 7 were bled at six (Day 1),seven (Day 7), or one (Days 9 through 15) time point(s), respectively.Whole blood samples were collected in a lithium heparin microtainer andprocessed to plasma by centrifugation. Plasma was frozen at −70° C.Pharmacokinetic analyses were performed on the individual animal plasmaconcentration versus time data for MGBG using WinNonlin (lineartrapezoidal rule for AUC calculations). Nominal dose values and samplingtimes were used for calculations. For Study Day 7, the reported valuesfor MGBG concentrations at time zero were used in the calculations ofAUC. Study Day 1 disposition parameters were not reported due toinsufficient terminal phase data to adequately characterize theseparameters. Following PO administration of MGBG on Study Day 7, plasmaPK disposition parameters were calculated on plasma concentrationsobtained following the second administered dose (T=12-192 h) using theWinNonlin default selection criteria for the selection of the Lambda Z,the elimination rate constant, upon which half-life, AUC_(INFobs), andCl/F_(obs), were based; inter-animal variability was noted.

Plasma samples collected from test article-treated animals on Day 1 andDay 7 were subjected to bioanalysis and confirmed systemic exposure tothe test article at all time points. Over the dose range evaluated,T_(max) values were dose-dependent and ranged from 3.33 to 14.0 h, andindicated absorption was slightly delayed on Study Day 7 compared toStudy Day 1. Systemic exposure (as assessed by C_(max) and AUC_(all))increased with increasing dose, and the increase in both parameters wasslightly less than dose-proportional at each evaluation interval.Repeat, twice-daily PO dosing of MGBG was associated with 3.77-, 4.03-,and 3.68-fold increases in mean AUC_(all) values compared to Study Day 1for the 20, 40, and 60 mg/kg/day dose groups, respectively. On Study Day7, evidence of dose-dependent dispositions for Cl/F_(obs) andelimination half-life were observed as mean parameter values forCl/F_(obs) and elimination half-life increased and decreased,respectively, with increasing dose levels.

Differences between the control group and the 60 mg/kg/day dose groupwere noted for a few hematology parameters (lower reticulocyte count andpercentage) and some serum chemistry parameters (e.g., osmolality andelectrolyte changes consistent with slight dehydration). However, thesechanges were not thought to be adverse as they did not coincide withother signs of frank toxicity and the serum chemistry changes weredemonstrated to be reversible. No gross or microscopic lesions wereobserved in test article-treated animals at terminal sacrifice, and nogross lesions were observed in test article-treated animals at recoverysacrifice.

Based on the findings of this exploratory study, the no observableadverse effect level (NOAEL) for MGBG administered by PO gavage twicedaily for seven consecutive days to male Sprague Dawley rats is 30mg/kg/dose (60 mg/kg/day).

TABLE 4 PO Dose AUC AUC₀₋₁₂ AUC₀₋₂₄ Day 1 C_(max) T_(max) (h * ng/ (h *ng/ (h * ng/ (mg/kg/day) (ng/mL) (h) mL) mL) mL) Cl/F_(obs) 20 124 10 NC773 1750 NC 40 175 7.33 NC 1380 2980 NC 60 296 3.33 NC 2170 4380 NC

TABLE 5 PO Dose AUC AUC₀₋₁₂ AUC₀₋₂₄ Day 7 C_(max) T_(max) (h * ng/ (h *ng/ (h * ng/ (mg/kg/day) (ng/mL) (h) mL) mL) mL) Cl/F_(obs) 20 117 147930 1030 2190 24.3 40 194 9.33 13700 1800 3690 28.2 60 337 7.33 171003130 6040 35.8

Allometric Scaling and Predicted Human Efficacy

Multi-species allometric scaling based on pharmacokinetic parametersdisclosed in Tables 2 and 3 was employed to calculate predictedpharmacokinetic parameters in humans according to methods known in theart. See, e.g., Ings R M, “Interspecies scaling and comparisons in drugdevelopment and toxicokinetics,” Xenobiotica, 1990 November;20(11):1201-31 and Khor, S P et al., “Dihydropyrimidine dehydrogenaseinactivation and 5-fluorouracil pharmacokinetics: allometric scaling ofanimal data, pharmacokinetics and toxicodynamics of 5-fluorouracil inhumans,” Cancer Chemother Pharmacol (1997) 39(3): 833-38. Expectedvalues are given below in Tables 6 and 7.

TABLE 6 CL V_(SS) IV t_(1/2) (h) (mL/min/kg) (L/kg) Based on 13.4 7.79.0 Mouse, Rat, Dog, Rhesus Based on 13.3 7.9 9.1 Mouse, Dog, Rhesus

TABLE 7 CL V_(SS) ORAL t_(1/2) (h) (mL/min/kg) (L/kg) Based on 23.3 21.042.4 Mouse, Rat, Dog, Rhesus Based on 23.0 20.9 41.6 Mouse, Dog, Rhesus

In both the murine carrageenan-induced paw edema and hyperalgesiamodels, the top efficacious dose of MGBG is 30 mg/kg PO BID (totaling 60mg/kg/day). Based upon this dosing paradigm in mice, at least twomethods to estimate the equivalent dosing in humans may be used.

The first method is based upon body surface area (BSA) normalization(described in Reagen-Shaw et al. (2007) FASEB J. 22, 659-661), as theauthors note that BSA correlates well across species for variousbiological parameters, including basal metabolic rate, blood volume,caloric expenditure, plasma protein levels, and renal function. Usingthis method, a 60 mg/kg/day dose in mice would convert to about 4.9mg/kg/day in humans.

The second method used to convert the efficacious 60 mg/kg/day dose inmice to an equivalent dose in humans was based more directly onallometric scaling. Data from an MGBG pharmacokinetic study consistingof a 10 mg/kg oral dose in mice was modeled in a simulation to determinethe theoretical AUCINF value for a dosing regimen of 30 mg/kg PO BID,which was 9050 h*ng/mL. Next, predicted human clearance values asdetermined by single- and multi-species allometric scaling were used toestimate doses likely to produce an exposure in humans (AUCINF) similarto that of the 60 mg/kg/day in mice. Using single-species allometricscaling and a range of predicted human clearance values, a humanequivalent dose would be in the range of 1.73 mg/kg/day to 4.51mg/kg/day. Using multi-species allometric scaling, the predicted humanequivalent dose is about 4.2 mg/kg/day.

In the murine carrageenan models, we also observed efficacy of MGBG atlower doses, including 3 mg/kg PO BID and 10 mg/kg PO BID, which wouldproportionally convert to human doses of ˜0.42 mg/kg/day and ˜1.2mg/kg/day.

The average body weight of a normal male human is often presumed to be70 kg. Thus, daily doses based on the predictions above could beestimated to range from about 25 mg/day to about 350 mg/day.

The proper dose depends, of course, on a number of factors. The patientmay weigh much more or much less, or be female, elderly, or juvenile,requiring a lower or higher dose. The patient may exhibit a drugmetabolic profile which might counsel for a lower or higher dose, suchas a low expression level or activity of metabolizing enzymes such ascytochromes P450 (CYPs). This low expression or activity level may bedue to a number of factors. Polymorphic expression of one or more CYPs(for example CYP2C19 and CYP2D6, though polymorphisms have beendescribed for nearly all the CYPs) is known to be responsible for somepopulations to be “deficient” as compared to the population at large,leading to a “poor metabolizer” phenotype, requiring a lower dose.Additionally, exposure to an infectious agent or xenobiotic may causerepression of CYP expression or inhibition of existing CYPs.Alternatively, the patient may be physically weak, injured, orimmunocompromised, all of which might counsel a lower dose. The patientmay be taking a number of other drugs which compete with metabolicsystems (including CYPs as discussed above) for disposal; this well-knowpolypharmaceutical effect may call for a lower dose. The dose alsodepends, as discussed above, on the condition and its severity. Theefficacious dose for one disease or clinical endpoint will notnecessarily be the same as the dose for another, and a severe, chronic,or otherwise serious case may call for a higher dose. However, a chroniccase may also call for a lower dose administered over a longer or evenindefinite period of time. All of these are discussed by way of exampleto illustrate the variability of ideal dosing; it is within the capacityof the skilled artisan to select an appropriate dosing range for adisease, population, or individual.

With these factors in mind, it should be clear that it is possible thatthe daily human dose may be as low as 1 mg/day, and as high as a 1g/day. In certain embodiments, the human dose may range: from 10 mg/dayto 500 mg/day, from 20 mg/day to 400 mg/day, or from 25 mg/day to 350mg/day. In further embodiments, the human dose may range: from 120mg/day to 350 mg/day, from 150 mg/day to 350 mg/day, from 200 mg/day to350 mg/day, or from 250 mg/day to 350 mg/day. In certain embodiments,the human dose may be any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15,20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 75, 80, 85, 90, 95, 100, 110,120, 125, 130, 140, 150, 160, 170, 175, 180, 190, 200, 210, 220, 225,230, 240, 250, 260, 270, 275, 280, 290, 300, 310, 320, 325, 330, 240 or350 mg/day.

In certain embodiments, the human dose may be any one of 275, 280, 285,290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 350, 355, 360,365, 370, or 375 mg/day. In one embodiment, the dose may be 275 mg/day.In another embodiment, the dose may be 300 mg/day. In anotherembodiment, the dose may be 305 mg/day. In another embodiment, the dosemay be 310 mg/day. In another embodiment, the dose may be 315 mg/day. Inanother embodiment, the dose may be 320 mg/day. In another embodiment,the dose may be 325 mg/day. In another embodiment, the dose may be 330mg/day. In another embodiment, the dose may be 335 mg/day. In anotherembodiment, the dose may be 340 mg/day. In another embodiment, the dosemay be 345 mg/day. In another embodiment, the dose may be 350 mg/day.

In certain embodiments, the human dose may be any one of 350, 375, 400,425, 450, 475, 500, 525, 550 or 600 mg/day. In one embodiment, the dosemay be 375 mg/day. In another embodiment, the dose may be 400 mg/day. Inanother embodiment, the dose may be 450 mg/day. In another embodiment,the dose may be 500 mg/day.

In certain embodiments, the human dose may be any one of 25, 50, 75,100, or 125 mg/day. In one embodiment, the dose may be 375 mg/day. Inanother embodiment, the dose may be 25 mg/day. In another embodiment,the dose may be 50 mg/day. In another embodiment, the dose may be 75mg/day. In another embodiment, the dose may be 100 mg/day. In anotherembodiment, the dose may be 125 mg/day.

In Vivo Carrageenan Tests

Carrageenan Paw Test for Edema and Hyperalgesia

Injection of carrageenan subcutaneously into the hind foot (paw) of arat or mouse induces robust inflammation and pain. The inflammatoryresponse begins 1-2 hrs post-carrageenan injection and persists for atleast five hours following inoculation. In addition, the animal'sinflamed hind paw is sensitive to noxious (hyperaglesia) or innocuous(allodynia) stimuli, compared to the contralateral hind paw. Compoundscan be evaluated in this model for anti-hyperalgesia andanti-inflammatory activity. A general increase in threshold or time torespond following drug administration suggests analgesic efficacy. Ageneral decrease in paw swelling following drug administration suggestsanti-inflammatory efficacy. It is possible that some compounds willaffect the inflamed paw and not affect the responses of thecontralateral paw.

Embodiments of the carrageenan foot edema test are performed withmaterials, reagents and procedures essentially as described by Winter,et al., (Proc. Soc. Exp. Biol. Med., 111, 544 (1962)). Prophylactic andtherapeutic embodiments have been developed, and are known in the art.The animals are evaluated for their responsiveness to noxious (pawpinch, plantar test) or innocuous (cold plate, von Frey filaments)stimuli. In the following protocol, mice were used.

Animals, compounds, and dosing. Healthy young male Swiss Webster mice inwhich weight variation of the mice will not exceed ±20% of the mean wereused for the study. Animals were divided into four groups of forty, andeach group was dosed by oral gavage with either MGBG (BID, 12 hoursapart at 30 mg/kg in 5 mL/kg normal saline), dexamethasone as positivecontrol (QD, 1 mg/kg in 5 mL/kg 0.5% methylcellulose), or saline vehicle(BID, 5 mL/kg). A fourth group served as naïve control (no carrageenan,no treatment). Treatment with MGBG took place on each of three daysprior to carrageenan, one hour prior to carrageenan, and 11 hourspost-carrageenan. Paw edema is developed by injecting carrageenan(Sigma: λ-carrageenan) subcutaneously in the subplantar region of theright paw of the mouse at a volume of 50 μl of 1% carrageenan (w/v) insaline. The contra-lateral paw (left paw) received the same volume (50ul) of saline and serve as control. Mice will be anesthetized usinglight dose of ketamine before carrageenan injection.

Paw Edema. Immediately before sub-plantar administration of carrageenanand after 2, 3, 5 and 24 hours post carrageenan, mouse paw volume wasmeasured using the plethysmometer (Ugo Basile). The assessment of edemawas expressed as the mean increase in paw volume relative to control.

Assessment of Paw Withdrawal Latency. Prior to sub-plantaradministration of carrageenan and after 0.5, 2, 3, 5, and 24 hourspost-carrageenan, the latency of withdrawal response was determined byplacing mice on a hot plate analgesia meter with surface temperaturemaintained at 51° C. A cut-off period of 30 s was maintained to avoidany thermal injury to paw. Immediately after testing, all paws wereimmersed in ice-cold water before returning to the cage. Paw withdrawallatency is calculated as Δt=right paw withdrawal−left paw withdrawal.

Serum, Plasma, and Histological Collection. Prior to first drug dose onday 0 and at peak disease times (5 and 24 hours post-carrageenanchallenge for serum, prior to first drug dose on day 0 and at conclusionof study), serum or plasma was collected from eight mice per group(each) and stored at −70° C. until cytokine level determination or MGBGdrug level determination. For serum collection, whole blood samples arecollected in a serum separator tube, processed by centrifugation andfrozen at −70° C. For drug level determination, whole blood samples arecollected in a lithium heparin microtainer, processed to plasma bycentrifugation and plasma frozen at −70° C. Additionally, paws arecollected and preserved in 10% formalin for histology.

Alternative Protocol. In an alternative embodiment of this assay, MGBGwas dosed PO, BID at 3, 10, and 30 mg/kg (with dexamethasone as positivecontrol, saline as negative, and a treatment/carrageenan-naïve group,n=16 each).

Results. MGBG was efficacious in reducing edema and hyperalgesia in theabove assay.

Carrageenan Air Pouch Model

Injection of air subcutaneously will induce the formation of aconnective tissue cavity lined with cells that resemble and functionlike a synovial lining. This method is commonly known as the Air PouchModel and is a useful animal model of inflammation that can be generatedin a relatively short period of time. The air pouch may be created bysubcutaneous (SQ) injection of a volume of sterile air in the dorsalneck region. The model may be used to test the efficacy and potency ofcompounds such as polyamine analogues and polyamine biosynthesisinhibitors such as MGBG in reducing various cellular and biochemicalindicators of inflammation when administered as an oral (PO) gavageadministration.

Animals, compounds, and dosing. Healthy male Swiss Webster mice of 7-9weeks of age and at least 30 g in weight were used. Animals were dividedinto six groups of thirty. Each group was administered 6 mL of airsubcutaneously as described below on days 4 and 7. Groups 1-5 wereadministered test article or control by oral gavage: either MGBG (atdose levels of 3, 10, and 30 mg/kg/dose BID, 12 hours apart, yieldingdaily doses of 6, 20, and 60 mg/kg/day), dexamethasone as positivecontrol (QD, 1 mg/kg in 10 mL/kg 0.5% methylcellulose), or vehicle (BID,10 mL/kg). The sixth group served as naïve control (no carrageenan, nodrug). The final doses on day 10 were given one hour prior to challengewith carrageenan. Groups 1-5 received 15 mg of carrageenan in 1 mLvehicle subcutaneously; group 6 received vehicle only. Tolerance of testcompound dosing was monitored in mice by daily visual inspections ofphysical and behavioral changes, such as examined changes in bodyposture, hair coat, activity, excreta, etc., and dosing reevaluated uponnegative findings. Mice are fed Purina rodent chow and water throughoutthe study.

Appropriate amounts of MGBG are made fresh for each administration(twice daily) by dissolving in appropriate volumes of normal saline toprepare PO gavage dosing formulations with concentrations of 0.3, 1.0,or 3.0 mg/ml. Similarly, an appropriate amount of dexamethasone isdissolved in a volume of 0.5% methylcellulose to prepare a doseformulation with a concentration of 0.1 mg/mL. For carrageenan, a 1.5%(weight/volume) solution of λ-carrageenan in 0.5% methylcellulose with0.025% Tween 20 is prepared the day prior to dosing. Carrageenan powderis gradually added to the 0.5% methylcellulose with 0.025% Tween 20solution (approximately 90% of the final volume) and the formulation isallowed to stir slowly overnight (avoiding bubbling and frothing). Thefollowing morning, the formulation is inspected to verify that asolution has been prepared, and if so, the formulation is brought up toits final volume.

Air pouch formation and maintenance: The mice were anesthetized byinhalation of isoflurane. The nape of the neck was shaved and cleaned bywiping the epidermis with 70% ethanol and subsequently with anappropriate topical disinfectant (i.e., Nolvasan, povidone, etc.). A23-gauge needle was attached to an appropriately sized syringe filledwith 6 mL of air and fitted with a 0.22 urn filter. Six mL of sterileair was then injected subcutaneously and the mouse returned to its cage,after dosing on Day 4 and again on Day 7 to reinflate the air pouches.On Day 7, the volume of air may be decreased to avoid over-inflation ofthe air pouch.

Carrageenan Challenge: One hour after the morning PO test articleadministration on Day 10, the surviving mice in each group wereanesthetized was be injected into the air pouch of each animal, and themouse will be returned to its cage.

Exudate, Plasma, and Histological Collection. The systemic exposure ofthe test article was evaluated as follows. Plasma samples were preparedand evaluated as follows. Two hundred μL whole blood was collectedretro-orbitally under isoflurane anaesthesia from Groups 1-6 at two timepoints, on Day 4 (all animals, Cohorts A, B, C; approximately half-waybetween the morning and evening dose) and on Day 10 (Cohort C; at 24hours after the morning MGBG dose). Samples were collected in lithiumheparin and processed to plasma by centrifugation, and frozen at −70° C.The plasma samples from the time point at 24 hours after the morningMGBG dose on Day 10 (Cohort C) were analyzed by LC/MS/MS for testarticle levels (approximately 60 samples).

Air pouch exudates were prepared and evaluated as follows. Cohorts of 10animals per group from all groups are sampled at one of three timepoints: prior to MGBG dosing and carrageenan injection on Day 10 (CohortA) and at approximately 6 and 24 hours (Cohorts Band C, respectively)after carrageenan injection on Day 10. Each dose group consisted of 3cohorts of 10 animals each. Starting with the first time point, a newcohort was sampled at each successive time point. Animals wereanaesthetized by CO₂ inhalation, then 2 mL of sterile PBS containing 10μM indomethacin is injected into the air pouch of each mouse in therespective cohorts at the time points indicated above. Following theinjection, the pouch was gently massaged for a few seconds. The pouchwas then opened with surgical scissors, and the exudate aspirated fromthe pouch with a plastic, disposable transfer pipette and placed in atest tube on ice. The weight of each test tube filled with exudate wasmeasured, and this value used to determine the exudate volume within thetube (minus each tube's weight) prior to centrifugation for 10 minutesat 1000×g. After centrifugation the supernatant and cell pellet weredivided and handled as follows.

The pouch exudate supernatant was divided into four aliquots A, B, C,and D, with 500 μL of supernatant allocated to Aliquots A, B, and C, andthe remaining exudate supernatant allocated to Aliquot D, as recoverablevolume allows. Aliquots were analyzed via LC MS/MS (test articleanalysis); multiplex assay or ELISA as appropriate (biomarker analysis).All aliquots were frozen at −70° C. Aliquot A may be analyzed for PGE2and 6-keto-PGF1alpha. Aliquot B may optionally be analyzed for any oneor more of osteopontin, IL-10, IL-12 (p70), MMP-9, TNFα, IL-1α, IL-1β,IL-6, MCP1, MCP3, MIP-1β, MIP-2, Rantes, IFNγ, IL-17, and CRP. Aliquot Cmay optionally be analyzed for IL-12 (p40), IFNα, IFNβ, IP10, MCP2,PGE2, 6-keto-PGF1alpha, and TGFβ1. Aliquot D may optionally be analyzedfor MGBG.

The cell pellet was gently resuspended in 1.5 mL of PBS, and aliquotedinto three aliquots of 0.5 mL each. One aliquot was analyzed for cellcount/differential analysis.

The pouch lining was collected and divided approximately in half. Thefirst half was preserved in 10% neutral buffered formalin for potentialhistology. The second half was flash frozen and stored at approximately−70° C., for potential analysis of osteopontin levels viaimmunohistochemistry.

The protocol may be varied according to methods known in the art.Additional tissue may be collected and/or weighed, and additionalsectioning, staining, and microscopic examination may be conducted.

MGBG was efficacious in this model, as shown by changes relative tocontrol indicative of reduced inflammation. It is expected that otherpolyamine analogs and polyamine biosynthesis inhibitors and compoundsdisclosed herein would be active as well.

In Vivo Murine Collagen-Induced Arthritis

Collagen-Induced Arthritis Models of Arthritis and Rheumatoid Arthritis

The collagen-induced arthritis (CIA) model is considered a suitablemodel for studying potential drugs active in human arthritis because ofthe many immunological and pathological similarities to human rheumatoidarthritis (RA), the involvement of localized major histocompatibility,complete class-II-restricted T helper lymphocyte activation, and thesimilarity of histological lesions. See, e.g., Rosloniec E F et al.,“Collagen-Induced Arthritis,” Current Protocols in Immunology, Unit 15.5(1993). See also the model using a monoclonal antibody to CD18 and VLA-4integrins described in Issekutz, A. C. et al., Immunology (1996) 88:569.Features of this CIA model that are similar to that found in RA patientsinclude, without limitation: erosion of cartilage and bone at jointmargins (as can be seen in radiographs), proliferative synovitis,symmetrical involvement of small and medium-sized peripheral joints inthe appendicular, but not the axial, skeleton. The following procedurewas followed to assess the efficacy of MGBG in the treatment ofarthritic diseases.

Animals and dosing. Inbred male DBA/1 mice (DBA/1OlaHsd, HarlanLaboratories), at least 7 weeks old, may be used in the followingcollagen-induced arthritis model. Twenty animals per compound or vehicleare assigned to the arthritis and saline groups, 4 to the control group.To induce an arthritic state, mice are anesthetized with isoflurane andgiven 1501 μl of bovine type II collagen in Freund's complete adjuvantinjections (day 0 and day 21). Mice are randomized by body weight intotreatment groups on study day 7. Treatment consists of 25 mg/kg MGBG,0.2 mg/kg dexamethasone as positive control, or saline as vehiclecontrol, all given as oral gavage beginning on study day 0 andcontinuing daily (PO, BID twice daily/12 hours apart). Twenty mice pergroup may be used, in which serum is collected from 15 animals, andplasma from five. Four additional animals serve as normal (untreated,non-arthritic) control group. The in-life portion of the study mayproceed for 35 days.

Compounds. MGBG solution may be made from the hydrated dihydrochloridesalt; other salts could be used, and in any case a salt/hydratecorrection factor should be implemented. Solid MGBG can be stored atroom temperature, but dose formulations should be made fresh for eachadministration. Dexamethasone is commercially available.

Data. On days 21-35, onset of arthritis typically occurs. During thistime clinical scores for paw edema and swelling were given for each ofthe paws (right front, left front, right rear, left rear). Plasma drawsare taken on days 0, 14, and 25 to assess pharmacokinetics, and blooddraws taken on days 0 and 28 for disease analysis. Edema is measured ondays 18-20, 22-27, and 29-34. Inflammation is assessed by infiltrationof inflammatory cells and edema. Post-euthanasia, terminal blood drawsare collected, heparinized, and frozen at −70° C. until analyzed forcytokines such as osteopontin, TNFalpha, IL-1, CRP, MCP1, MIP-lbeta,RANTES, IFNgamma, TGFbeta, IP-10, IL-17, and MMP9. Fore and hind pawsand knees are collected, and following 1-2 days in fixative and then 4-5days in decalcifier, processed, embedded, sectioned and stained withtoluidine blue for histological analysis. Bone resorption is quantifiedby presence of osteoclasts, defects in or loss of medullary trabecularor cortical bone. Cartilage damage is assessed by examining the severityand spread of chondrocyte loss and collagen disruption. Pannus tissueformation and the severity and spread of other evidence of destructionof joint architecture is followed.

Statistical Analysis. Clinical data for paw scores (means for animal)are analyzed by determining the area under the dosing curve (AUC) fordays 1-15. For calculation of AUC, the daily mean scores for each mouseare entered into Microsoft Excel and the area between the treatment daysafter the onset of disease to the termination day is computed. Means foreach group are determined and % inhibition from arthritis controlscalculated by comparing values for treated and normal animals. Pawscores and histologic parameters (mean±SE) for each group are analyzedfor differences using a Student's t-test with significance set at p0.05. Percent inhibition of histologic parameters and AUC is calculatedas [(mean disease control−mean normal)−(mean treated−meannormal)]/[[(mean disease control−mean normal)·(mean treated−meannormal)]·100.

Expectations. It is expected that MGBG, as well as other polyamineanalogs and polyamine biosynthesis inhibitors and compounds disclosedherein, will be efficacious in this model as shown by prevention of,reduction of, or delay of onset of arthritic symptoms as discussedabove, reduced inflammation and related measures as discussed above,reduced measures of pain, and other related sequelae. The protocolsabove may be varied according to methods known in the art.

Additional In Vivo Models of Therapeutic Efficacy

The following models, presented by way of example, may be used toevaluate compounds disclosed herein for efficacy in the treatment of anumber of diseases and indications. It is within the capacity of oneskilled in the art to modify these models to suit the needs of thestudy. Additionally, those skilled in the art will be familiar withadditional models of disease which may be employed. It is expected thatMGBG, as well as other polyamine analogs and polyamine biosynthesisinhibitors and compounds disclosed herein, will be efficacious in thesemodels.

Neuropathy and Neuropathic Pain Models

Bennett Model of Neuropathic Pain:

A peripheral mononeuropathy is produced in adult rats by placing looselyconstrictive ligatures around the common sciatic nerve. Thepostoperative behavior of these rats indicates that hyperalgesia,allodynia and, possibly, spontaneous pain (or dysesthesia) wereproduced. Hyperalgesic responses to noxious radiant heat are typicallyevident on the second postoperative day and lasted for over 2 months.Hyperalgesic responses to chemogenic pain were also present. Thepresence of allodynia may be inferred from nocifensive responses evokedby standing on an innocuous, chilled metal floor or by innocuousmechanical stimulation (e.g., with von Frey filaments), and by the rats'persistence in holding the hind paw in a guarded position. The presenceof spontaneous pain is suggested by a suppression of appetite and by thefrequent occurrence of apparently spontaneous nocifensive responses. Theaffected hind paw is typically abnormally warm or cool in aboutone-third of the rats. About one-half of the rats develop grosslyovergrown claws on the affected side. In compound efficacy models, testcompound is typically delivered prior to stimulation and vehicle servesas control. Experiments with this animal model may advance understandingof the neural mechanisms of neuropathic pain disorders in humans.Bennett G J, Xie Y K, 1988 “A peripheral mononeuropathy in rat thatproduces disorders of pain sensation like those seen in man.” Pain,April; 33(1):87-107 (PMID: 2837713).

Chung Model of Neuropathic Pain

Since its introduction in 1992, the spinal nerve ligation (SNL) model ofneuropathic pain has been widely used for various investigative works onneuropathic pain mechanisms as well as in screening tests for thedevelopment of new analgesic drugs. This model was developed by tightlyligating one (L5) or two (L5 and L6) segmental spinal nerves in the rat.The operation results in long-lasting behavioral signs of mechanicalallodynia, heat hyperalgesia, cold allodynia, and ongoing pain. In theprocess of widespread usage, many different variations of the SNL modelhave been produced, either intentionally or unintentionally, bydifferent investigators. Although the factors that cause thesevariations themselves are interesting and important topics to bestudied, the pain mechanisms involved in these variations are likelydifferent from the original model. The method for producing the spinalnerve ligation model that will minimally induce potential factors thatmay contribute to these variations is described in detail in Chung J M,Kim H K, and Chung K, “Segmental spinal nerve ligation model ofneuropathic pain,” Methods Mol Med.; 2004 99:35-45 (PMID: 15131327).

Chung Model in NHP

In a model of painful neuropathy in the primate (Macaca fascicularis), aneuropathic state is induced by tight ligation of the L7 spinal nerve,just distal to the L7 dorsal root ganglion. Sensory testing may be doneon the ventral surface of the foot, a region that includes the L7dermatome. Within 1 week following surgery, primates typically develop amarked sensitivity to mechanical stimulation (e.g., with von Freyhairs), indicating the presence of mechanical allodynia. Increasedsensitivity to mechanical stimulation is sometimes also observed on thecontralateral side. The threshold for withdrawal to a heat stimulusdecreases, indicating the presence of heat hyperalgesia. Presentation ofvarious cooling stimuli, such as acetone and cold water baths, indicatesthat cold allodynia also develops. Observed behavioral phenomena aresimilar to those seen in humans diagnosed with peripheral neuropathicpain. Thus, the model is useful for assessing a number of parametersrelevant to human neuropathy and neuropathic pain disorders, and forevaluating the efficacy of drug candidates as treatments for relateddisorders. See, e.g., Carlton S M et al., “Behavioral manifestations ofan experimental model for peripheral neuropathy produced by spinal nerveligation in the primate,” Pain 1994 February; 56(2):155-66 (PMID:8008406).

Tactile Allodynia Assessment with Von Frey Filaments

The following quantitative allodynia assessment technique may bemodified to measure tactile allodynia in any of the various animalmodels of neuropathic pain. The following summary is given by way ofexample and refers to a rat surgical neuropathy model whereinnocifensive behaviors are evoked by light touch to the paw. Employingvon Frey hairs from 0.41 to 15.1 g, the percent response at eachstimulus intensity may first be characterized. A smooth log-linearrelationship is typically observed. Additionally or alternatively, aparadigm using stimulus oscillation around the response threshold may beemployed, which allows more rapid, efficient measurements. Correlationcoefficient between the two methods is typically high. In neuropathicrats, good intra- and inter-observer reproducibility is found for theup-down paradigm; some variability may be seen in normal rats,attributable to extensive testing. The fact that thresholds in a sizablegroup of neuropathic rats show insignificant variability over 20 days,and after 50 days, 61% still met strict neuropathy criteria (usingsurvival analysis), indicates that threshold measurement using theup-down paradigm, in combination with the neuropathic pain model,represents a powerful tool for analyzing the effects of manipulations ofthe neuropathic pain state. See, e.g., Chaplan S R et al., “Quantitativeassessment of tactile allodynia in the rat paw.” J Neurosci Methods,1994 July; 53(1):55-63 (PMID: 7990513).

Hargreaves Method of Assessing Thermal Nociception

Alternatively, a method to measure cutaneous hyperalgesia to thermalstimulation in unrestrained animals has been described. The testingparadigm uses an automated detection of the behavioral end-point;repeated testing does not contribute to the development of the observedhyperalgesia. Carrageenan-induced inflammation results in significantlyshorter paw withdrawal latencies as compared to saline-treated paws andthese latency changes corresponded to a decreased thermal nociceptivethreshold. This sensitive thermal method detects dose-relatedhyperalgesia and its blockade by test compounds and allows for themeasurement of other behavioral parameters in addition to thenociceptive threshold. See, e.g., Hargreaves K, et al., “A new andsensitive method for measuring thermal nociception in cutaneoushyperalgesia,” Pain, 1988 January; 32(1):77-88 (PMID: 3340425).

HIV Dementia Models

HIV Dementia Macaque Models

Human Immunodeficiency virus (HIV), the virus that causes acquiredimmunodeficiency syndrome (AIDS), also manifests neurologicalcomplications. HIV-associated dementia (HAD) is the most severe form ofHIV-induced neurocognitive disorders. HIV encephalitis (HIVE), thepathological correlate of HAD, is characterized by the formation ofmultinucleated giant cells and microglial nodules, astrocytosis, andneuronal damage and loss. Pathological evaluation of HAD diseaseprogression in humans is not possible, with the only data collectedbeing from individuals who have succumbed to the disorder, a snap shotof end-stage disease at best. Therefore, pertinent animal models havebeen developed to alleviate this gap of knowledge in the field ofneurovirology and neuroinflammation. In general, the most widely usedanimal models are the simian immunodeficiency virus (SIV) and thechimeric simian/human immunodeficiency virus (SHIV) macaque modelsystems. Although both SIV and SHIV model systems are able to potentiateneuroinvasion and the concomitant neuropathology similar to that seen inthe human syndromes, the innate differences between the two in diseasepathogenesis and progression make for two separate, yet effective,systems for the study of HIV-associated neuropathology. For a thoroughcomparison of these two models, see Williams R et al., “Nonhuman primatemodels of NeuroAlDS,” J Neurovirol. 2008 August; 14(4):292-300 (PMID:18780230). An exemplary SIV model is given below.

Simian Immunodeficiency Virus (SIV) Model

The neuropathogenesis of human immunodeficiency virus (HIV)-associateddementia has remained elusive, despite identification of HIV as thecausal agent. Although a number of contributing factors have beenidentified, the series of events that culminate in motor and cognitiveimpairments after HIV infection of the central nervous system (CNS) arestill not known. Rhesus monkeys infected with simian immunodeficiencyvirus (SIV) manifest immunosuppression and CNS disease that ispathologically [L. R. Sharer et al. (1991) J. Med. Primatol. 20,211-217] and behaviorally [E. A. Murray et al. (1992) Science 255,1246-1249] similar to humans. The SIV model of HIV-associated dementia(HAD, HIV dementia, AIDS dementia, AIDS dementia complex, ADC,neuroAIDS, HIV-associated neurodegeneration, HAND) is widely recognizedas a highly relevant model in which to investigate neuropathogenesis.With better understanding of neuropathogenesis comes the opportunity tointerrupt progression and to design better treatments for HAD. Thisbecomes increasingly important as patients live longer yet still harborHIV-infected cells in the CNS. The use of the SIV model has allowed theidentification of neurochemical markers of neuropathogenesis importantnot only for HAD, but also for other inflammatory neurological diseases.

The SIV model offers an ideal opportunity to investigate theneuropathogenesis of HAD. SIV is genetically, antigenically, andmorphologically similar to HIV. In the CNS, SIV predominantly infectsMG/MP, whereas it does not productively infect neurons. Rhesus monkeys(Macaca mulatta) inoculated with SIV display neuropathological featuressimilar to those seen with HAD, namely, multinucleated giant cellscontaining SIV, white matter lesions, and astrocytosis, although thereare some pathological differences. The pattern of disease progression inmonkeys resembles that in humans, with an initial burst of viralreplication, followed by a latency period before development ofimmunosuppression, opportunistic infections, and death. The time courseof disease progression is more rapid than that observed in humans, thusexpediting experimental analysis. The premier advantage of the SIV modelfor the study of HAD is the opportunity to investigate neurochemical andneuropathological changes in relation to the onset of behavioralimpairments in a nonhuman primate with a behavioral repertoire close tohumans. Such investigations are accomplished by killing animals atvarious points during disease progression, especially at early stages.An additional advantage of the SIV model is the elimination of theconfounding factor of treatment of HIV-infected individuals at end-stagedisease. In short, the SIV model lends itself to probing the temporal,and ultimately causal, sequence of CNS changes induced by infection todetermine the neuropathogenesis of HAD. The neuropathological correlatesof impairments uncovered by this model have utility for HAD and forother neurological diseases. See, e.g., Rausch D M et al., “TheSIV-infected rhesus monkey model for HIV-associated dementia andimplications for neurological diseases,” J Leukoc Biol. 1999 April;65(4):466-74 (PMID: 10204575).

Measuring HIV Dementia and HIV Encephalopathy in SIV Models

A battery of tasks may be chosen by the skilled investigator to assessencephalopathy and dementia in the SIV model. The tasks may be selected,for example, for their breadth in capturing neuropsychologicalimpairments, including those described in HIV-infected humans, and fortheir ability to target neural substrates well characterized in primatelesioning experiments and implicated in HAD.

HIV Dementia

For example, in Rausch et el., above, the tasks assessed cognitive andmotor function, and included: (1) delayed matching-to-sample with newstimuli on every trial to test visual recognition memory; (2) delayedmatching-to-sample with two repeatedly used stimuli to test recentmemory, (3) a visual discrimination learning and retention task to teststimulus-response association, and (4) a spatial scene learning task,which measured long-term memory for spatial locations. The motor taskassessed the ability of each monkey to retrieve food from a rotatingtable (via the turntable speed at which animals successfully retrievedfood in 50% of trials).

In one paradigm, a cohort of animals is trained on the battery of tasks,inoculated with SIV (optionally one or more isolated or molecularlycloned strains selected for properties such as neurovirulence), and thenevaluated on the tasks to detect changes in performance over a definedperiod. Alternatively, a separate cohort of animals is inoculated firstand then trained and evaluated for their ability to learn the tasks.Varying the timing of SIV inoculation relative to neurobehavioraltesting distinguishes between acquisition and retention of tasks.Infected animals may be considered impaired, for example, when theirscores were more than two standard deviations from the mean score ofuninfected control animals, which are sham-inoculated. While animals arealive, their clinical status, blood, and CSF may be assayed periodicallyto determine disease progression, immune status, and viral load.Neurochemistry and neuropathology may be performed at death. The range,extent, and variability of the cognitive and motor impairments resemblefindings with HIV-infected humans.

HIV Encephalopathy

An alternative model of HIV encephalopathy allows a more focused inquiryinto the pathology of HIV infection in the brain. Following inoculationwith SIV and development of moderate to severe encephalitis, viral loadmay be measured in cerebrospinal fluid (CSF) examined longitudinally toonset of AIDS and in brain tissue at necropsy to examine therelationship of systemic and central nervous system (CNS) viralreplication to the development of encephalitis. Persistent high levelsof CSF viral RNA after the acute phase of infection correlate with thedevelopment of encephalitis, and the level of both viral RNA and antigenin the brain correlate with the severity of the CNS lesions. Incontrast, plasma levels of viral RNA do not correlate to the developmentor severity of enceplalitis. Thus, CSF viral load measurements in thepostacute phase of SIV infection serve as a marker for encephalitis andCNS viral replication, an effective way to measure the efficacy of testcompounds in preventing or attenuating HIV encephalitis and relateddisorders. See, e.g., Zink M C et al., “High viral load in thecerebrospinal fluid and brain correlates with severity of simianimmunodeficiency virus encephalitis,”: J Virol. 1999 December;73(12):10480-8 (PMID: 10559366).

Additionally, metabolic markers such as NAA/Cr may also be used toquantitate disease severity in SIV/macaque models of encephalitis. Invivo MR spectroscopy (MRS) studies have shown reductions in NAA/Crlevels in human patients with severe neurocognitive deficits due to AIDSdementia complex (ADC), also known as neuroAIDS. High-field proton MRSwas performed on extracted metabolites from frontal cortex tissuesamples of 29 rhesus macaques (6 healthy, 23 moribund with AIDS).Neuropathologic determination of encephalitis severity for each animalwas completed and was found to correlate with NAA/Cr levels. Decreasesin Glu/Cr and GABA/Cr may indicate that both excitatory and inhibitoryneurons are affected. Highly significant correlations between NAA/Cr,Glu/Cr, and GABA/Cr were observed. These neuronal metabolites were alsodecreased in the absence of classical SIV encephalitis (SIVE). Thesestudies indicate that metabolic markers may serve as indicators ofdisease progression, and the efficacy of treatment at preventing ordelaying the development of NauroAIDS. See Lentz M R et al., “Metabolicmarkers of neuronal injury correlate with SIV CNS disease severity andinoculum in the macaque model of neuroAIDS,” Magn Reson Med. 2008 March;59(3):475-84 (PMID: 18306400).

FIV Model of HIV Dementia

Feline immunodeficiency virus (FIV) is a neurotropic lentivirus thatproduces a protracted state of immunodeficiency and encephalopathy inthe cat. Recent evidence has shown several similarities to the naturalprogression of human immunodeficiency virus infection (HIV-1) associateddegenerative effects on the central and peripheral nervous systems.Similar to HIV-1, FIV-induced encephalopathy neurovirulence is straindependent, results in progressive immunodeficiency and increasing earlyperipheral but not brain viral load, preferentially affects thedeveloping nervous system, produces quantifiable behavioral andneurophysiological impairment that is not directly linked to neuronalinfectivity, and induces neuronal injury and loss both in vivo and invitro. FIV has the added benefit of being non-communicable to humanresearchers. For these reasons, the FIV model is useful as a platformfor assessing the efficacy of compounds in the treatment of HIV dementiaand related disorders. See, e.g., Podell M et al., “The feline model ofneuroAIDS: understanding the progression towards AIDS dementia,” JPsychopharmacol. 2000; 14(3):205-13 (PMID: 11106298).

Inflammatory and Autoimmune Models

Contact Dermatitis and Related Disorders

Contact hypersensitivity is a simple delayed type hypersensitivity invivo assay of cell-mediated immune function, which can be used to assesspotential therapeutic efficacy in a number of disorders having aninflammatory and/or autoimmune component. Such diseases include contactdermatitis, atopic dermatitis, psoriasis, allergic dermatitis, anddermal irritation. Compounds may be topically applied, optionally in atopical formulation, or may be delivered by a non-topical (e.g., oral,IV, etc) route.

Murine Model

In one procedure, cutaneous exposure to exogenous haptens gives rise toa delayed type hypersensitivity reaction which is measured andquantitated. Contact sensitivity involves an initial sensitizing phasefollowed by an elicitation phase. The elicitation phase occurs when theT lymphocytes encounter an antigen to which they have had previouscontact. Swelling and inflammation occur, making this an excellent modelof human allergic contact dermatitis. Murine models also typically havethe additional benefit of being economical to run. A suitable procedureis described in detail in Gaspari A A and Katz S I, “ContactHypersensitivity,” Current Protocols in Immunology, Unit 4.2, John Wiley& Sons, Inc. (1994). See also Grabbe S and Schwarz T, “Immunoregulatorymechanisms involved in elicitation of allergic contacthypersensitivity,” Immun. Today 19 (1): 37-44 (1998).

Porcine Model

The choice of animal can be important in dematological studies intendedto predict human response. For this reason, pigs and in particularminipigs are favored due to the similarities between human and pig skin(particularly follicular density). See, for example, an exemplary modelin Bilski A J and Thomson D S, “Allergic contact dermatitis in thedomestic pig. A new model for evaluating the topical anti-inflammatoryactivity of drugs and their formulations,” Br J Dermatol, 1984 July; 111Suppl 27:143 (PMID: 6743545).

Hairless Guinea Pig Model

Allergic and irritant contact reactions have also been evaluated in therecently identified hairless guinea pig, Crl:IAF(HA)BR, a mutant fromthe Hartley strain. The irritant contact dermatitis may be induced bycroton oil, 2,4-dinitrochlorobenzene (DNCB), or anthralin. Both hairlessand hairy guinea pigs develop similar reactions to these chemicals.Photoallergic contact sensitization may be also induced withtetrachlorosalicylanilide (TCSA), or with cyclophosphamide beforesensitization with tribromosalicylanilide (TBS). Cutaneous changes areobserved macro- and microscopically according to methods known in theart. Thus, hairless guinea pigs can be used as animal models forassessment of test compounds in the treatment of immunologic andnonimmunologic contact reactions and related disorders. See, e.g.,Miyauchi H and Horio T, “A new animal model for contact dermatitis: thehairless guinea pig,” J Dermatol. 1992 March; 19(3):140-5(PMID:1640019).

Simple dermal irritation may also be studied in hairless guinea-pigs. Inan exemplary model, test compounds are delivered in one or more topicalformulations for 30 min daily exposure for 4 days. Scoring is performeddaily; evaporimetry (total epidermal water loss (TEWL)), hydration andcolorimetry are measured at baseline (day 0) in the middle and at theend of treatment. Test compounds are applied twice daily. See, e.g.,Andersen F et al., “The hairless guinea-pig as a model for treatment ofcumulative irritation in humans,” Skin Res Technol. 2006 February;12(1):60-7 (PMID: 16420540).

Psoriasis Murine Chimera Model

Additionally, the compounds disclosed herein can be tested in animalmodels for psoriasis-like diseases. Research into the cause andpathophysiological mechanisms underlying expression of psoriatric skinlesions has been hampered by lack of an appropriate animal model forthis common and enigmatic cutaneous disease. One suitable model is thehuman skin/scid mouse chimera prepared as described by Nickoloff B J etal., “Severe combined immunodeficiency mouse and human psoriatic skinchimeras. Validation of a new animal model,” Am J Pathol., 1995 March;146(3): 580-8 (PMID: 7887440). The methods described thereincharacterize normal skin, pre-psoriatic skin, and psoriatic plaque skinsamples transplanted onto severe combined immunodeficiency mice. Eithernormal, prepsoriatic, or psoriatic plaque keratome skin samples aretransplanted onto severe combined immunodeficiency mice reliably withhigh rates of graft survival (>85%) and with reproducible changesconsistently observed over prolonged periods of engraftment. Aftertransplantation, by clinical assessment and routine light microscopy,normal skin remains essentially normal whereas pre-psoriatic skin becamethicker, and psoriatic plaque skin retains its characteristicplaque-type elevation and scale. By using a panel of antibodies andimmunohistochemical analysis, the overall phenotype of human cell types(including immunocytes) that persisted in the transplanted skin wasremarkably similar to the immunophenotype of pretransplanted skinsamples. Additionally, clearly recognized interface zones between humanand murine skin within the epidermal and dermal compartments can beidentified by routine microscopy and immunostaining, with focal areas ofchimerism. The many similarities between pre- and post-transplantedhuman samples of normal and psoriatic skin that are grafted onto severecombined immunodeficiency mice make this animal model appropriate foruse in evaluating test compounds for efficacy in treating psoriasis andrelated disorders.

Psoriasis Murine scid/scid Model

Alternatively, the compounds disclosed herein can be tested in thescid/scid mouse model described by Schön M P et al., “Murinepsoriasis-like disorder induced by naive CD4+ T cells,” Nat Med., 1997February; 3(2):183-8 (PMID: 9018237). In this model, reconstitution ofscid/scid mice with minor histocompatibility mismatched naive CD4+ Tlymphocytes results in skin alterations that strikingly resemble humanpsoriasis clinically, histopathologically and in cytokine expression.

Asthma

Compounds may additionally be evaluated for efficacy in the treatment ofasthma and related pulmonary disorders. In one murine model of asthma,wild-type control [C57BL/6J, (+/+)] and ICAM-1 (intercellular adhesionmolecule-1) knockout [C57BL/6J-ICAM-1, (−/−)] mice are sensitized toovalbumin (OVA), and challenged with OVA delivered by aerosol (OVA-OVA)to induce a phenotype consistent with an asthmatic response. Bronchialresponsiveness to methacholine and counts of cell numbers andmeasurements of eosinophil content and cytokine levels inbronchoalveolar lavage fluid (BALF) may be measured. Additionally,lymphocyte proliferation in response to antigen, eosinophil migrationinto the airways, and the development of airway hyperreactivity (AHR) inallergen-sensitized and -challenged mice may all be measures in vivo orex vivio according to methods known in the art. See Wolyniec W W et al.,“Reduction of antigen-induced airway hyperreactivity and eosinophilia inICAM-1-deficient mice,” Am J Respir Cell Mol Biol., 1998 June;18(6):777-85 (PMID: 9618382).

Inflammatory Bowel Disease, Crohn's Disease, and Ulcerative Colitis

The compounds disclosed herein can also be evaluated for activity inanimal models of inflammatory bowel disease, Crohn's disease, andulcerative colitis. The protocol described by Scheiffele F, Fuss I J,“Induction of TNBS colitis in mice,” Curr Protoc Immunol, 2002 August;Chapter 15:Unit 15.19 (PMID: 18432874), is one of several that have beenused to study the immunopathogenesis of these diseases. The modelemploys the use of 2,4,6-trinitrobenzenesulfonic acid (TNBS), whichinduces severe colonic inflammation when administered intrarectally inSJL/J mice. The colitis which results from this procedure presentsclinical and histopathological findings that resemble those seen inCrohn's disease. Scheifflele and Fuss discuss the critical parametersneeded for successful induction of TNBS-colitis as well methods formonitoring and grading disease levels, and give a support protocol forisolating lamina propria mononuclear cells from mouse colons. See alsoMorris G P et al. “Hapten-induced model of chronic inflammation andulceration in the rat colon,” Gastroenterology, 1989 March;96(3):795-803 (PMID: 2914642), describing the original rat model ofchronic colonic inflammation by the intraluminal instillation of asolution containing a “barrier breaker” (e.g., 0.25 ml of 50% ethanol)and a hapten (e.g., TNBS, 5-30 mg) At a dose of 30 mg,trinitrobenzenesulfonic acid/ethanol-induced ulceration and markedthickening of the bowel wall persisted for at least 8 weeks.Histologically, the inflammatory response included mucosal andsubmucosal infiltration by polymorphonuclear leukocytes, macrophages,lymphocytes, connective tissue mast cells, and fibroblasts. Granulomas(3 wk after induction of inflammation), Langhan's-type giant cells,segmental ulceration and inflammation. The characteristics andrelatively long duration of inflammation and ulceration induced in thesemodels afford an opportunity to study the pathophysiology of colonicinflammatory disease in a specifically controlled fashion, and toevaluate new treatments potentially applicable to inflammatory boweldisease in humans.

Examplary Oral Pharmaceutical Formulations

The following are examples of compositions which may be used to orallydeliver compounds disclosed herein as a capsule.

A solid form of a compound of Formula VI may be passed through one ormore sieve screens to produce a consistent particle size. Excipients,too, may be passed through a sieve. Appropriate weights of compounds,sufficient to achieve the target dosage per capsule, may be measured andadded to a mixing container or apparatus, and the blend is then mixeduntil uniform. Blend uniformity may be done by, for example, sampling 3points within the container (top, middle, and bottom) and testing eachsample for potency. A test result of 95-105% of target, with an RSD of5%, would be considered ideal; optionally, additional blend time may beallowed to achieve a uniform blend. Upon acceptable blend uniformityresults, a measured aliquot of this stock formulation may be separatedto manufacture the lower strengths. Magnesium stearate may be passedthrough a sieve, collected, weighed, added to the blender as alubricant, and mixed until dispersed. The final blend is weighed andreconciled. Capsules may then be opened and blended materials flood fedinto the body of the capsules using a spatula. Capsules in trays may betamped to settle the blend in each capsule to assure uniform target fillweight, then sealed by combining the filled bodies with the caps.

COMPOSITION EXAMPLE 1

300 mg Capsule: Total fill weight of capsule is 500 mg, not includingcapsule weight. Target compound dosage is 300 mg per capsule, but may beadjusted to account for the weight of counterions and/or solvates ifgiven as a salt or solvated polymorph thereof. In such a case the weightof the other excipients, typically the filler, is reduced.

Ingredient Quantity per Capsule, mg MGBG 300.00 Lactose monohydrate179.00 Silicon dioxide 3.00 Crospovidone 15.00 Magnesium stearate(vegetable grade) 3.00

COMPOSITION EXAMPLE 2

150 mg Capsule: Total fill weight of capsule is 300 mg, not includingcapsule weight. Target compound dosage is 150 mg per capsule, but may beadjusted to account for the weight of counterions and/or solvates ifgiven as a salt or solvated polymorph thereof. In such a case the weightof the other excipients, typically the filler, is reduced.

Ingredient Quantity per Capsule, mg MGBG 150 Microcrystalline cellulose(MCC) 147 Magnesium stearate (vegetable grade) 3

All references cited herein are incorporated by reference as if writtenherein in their entireties. From the foregoing description, one skilledin the art can easily ascertain the essential characteristics of thisinvention, and without departing from the spirit and scope thereof, canmake various changes and modifications of the invention to adapt it tovarious usages and conditions.

1. An oral pharmaceutical composition, comprising MGBG together with atleast one oral pharmaceutically acceptable excipient, which yields atherapeutically effective systemic plasma MGBG level for at least a12-hour period when orally administered to a subject.
 2. The oralpharmaceutical composition as recited in claim 1, which yields atherapeutically effective systemic plasma MGBG level for at least a24-hour period when orally administered to a subject.
 3. The oralpharmaceutical composition as recited in claim 1, wherein the plasmalevel of MGBG is at least 75% of the peak plasma concentration for 4 ormore hours.
 4. The oral pharmaceutical composition as recited in claim1, having an oral bioavailability of at least 20%.
 5. The oralpharmaceutical composition as recited in claim 4, having an oralbioavailability of at least 30%.
 6. The oral pharmaceutical compositionas recited in claim 5, having an oral bioavailability of at least 35%.7. The oral pharmaceutical composition as recited in claim 6, whichyields a therapeutically effective plasma level of MGBG for at least a24 hour period in the subject with once-daily dosing.
 8. The oralpharmaceutical composition as recited in claim 1, having a half life ofat least 12 hours.
 9. The oral pharmaceutical composition as recited inclaim 8, having a half life of at least 18 hours.
 10. The oralpharmaceutical composition as recited in claim 1, which does not havesubstantially dose-limiting side effects.
 11. The oral pharmaceuticalcomposition as recited in claim 10, wherein said side effects aregastrointestinal.
 12. The oral pharmaceutical composition as recited inclaim 11, wherein said gastrointestinal side effects are chosen fromnausea, vomiting, diarrhea, abdominal pain, oral mucositis, oralulceration, pharyngitis, stomatitis, and gastrointestinal ulceration.13. The oral pharmaceutical composition as recited in claim 11, whereinsaid gastrointestinal side effects are chosen from inhibition ofgastrointestinal mucosal proliferation, inhibition of migration ofdeveloping epithelial lumen cells, and inhibition of differentiation ofstem or progenitor cells into epithelial lumen cells.
 14. The oralpharmaceutical composition as recited in claim 10, wherein said toxicside effects are chosen from thrombocytopenia, leukopenia, phlebitis,laryngitis, cellulitis, dermatitis, and hypoglycemia.
 15. The oralpharmaceutical composition as recited in claim 1, comprising about 25 mgto about 350 mg of MGBG.
 16. The oral pharmaceutical composition asrecited in claim 1, comprising about 250 mg to about 350 mg of MGBG. 17.The oral pharmaceutical composition as recited in claim 16, which yieldsa therapeutically effective plasma level of MGBG for at least a 24 hourperiod in the subject with once-daily dosing.
 18. The oralpharmaceutical composition as recited in claim 17 which does not havesubstantially dose-limiting side effects.
 19. The oral pharmaceuticalcomposition as recited in claim 1, formulated as a tablet or capsule.